Compositions, kits, and methods for effecting adenine nucleotide modulation of DNA mismatch recognition proteins

ABSTRACT

Compositions, and products comprising a MutS homolog which binds to a mismatched region of a duplex DNA molecule in the presence of ADP are provided, as are methods of binding MutS homologs to mismatched DNA in the presence of ADP. The use of MutL homolog derivatives in combination with MutS homologs is also included. Nonhuman mammals which are nullizygous for both Msh2 and p53 are also provided, as are methods of making and using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. Non-Provisional PatentApplication Ser. No. 09/143,571, filed Aug. 28, 1998, and is entitled topriority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Application60/093,935, filed Jul. 23, 1998, to U.S. Provisional Application No.60/066,977, filed Nov. 28, 1997, and to U.S. Provisional Application No.60/057,136, filed Aug. 28, 1997.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH AND DEVELOPMENT

[0002] This research was supported in part by U.S. Government funds (NIHgrants numbers CA56542 and CA67007 and NRSA grant CA73134), and the U.S.Government may therefore have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The field of the invention is DNA mismatch protein binding,including animals useful as models for tumorigenesis, apoptosis, andaging.

[0004] DNA Mismatch Repair

[0005] The most widely accepted model for DNA post-replication mismatchrepair is based largely on the model of the DNA adenine methylation(Dam)-Instructed pathway of Escherichia coli proposed by Modrich (1986,Basic Life Sci. 38:303-310; Modrich, 1987, Ann. Rev. Biochem.56:435-466; Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich, 1991,Annu. Rev. Genet. 25:229-253; Modrich et al., 1996, Annu. Rev. Biochem.65:101-133). According to this model, the MutS protein recognizes andbinds mismatched nucleotides resulting from polymerase misincorporationerrors to form a MutS-DNA product (Su et al., 1986, Proc. Natl. Acad.Sci., USA 83:5057-5061; Su et al., 1988, J. Biol. Chem. 263:6829-6835).MutS mismatch binding is followed by the interaction of MutL proteinwith the MutS-DNA product (Grilley et al., 1990, Mutat. Res.236:253-267), which accelerates ATP-dependent translocation of theMutS-MutL complex (Allen et al., 1997, EMBO J. 16:4467-4476) to ahemimethylated GATC Dam site to which MutH protein is bound (Welsh etal., 1987, J. Biol. Chem. 262:15624-15629; Au et al., 1992, J. Biol.Chem. 267:12142-12148). The MutS-MutL complex stimulates an intrinsicendonuclease activity of MutH protein, which cleaves the non-methylated(i.e. more recently replicated) DNA strand (Welsh et al., 1987, J. Biol.Chem. 262:15624-15629; Lahue et al., 1987, Proc. Natl. Acad. Sci. USA84:1482-1486; Su et al., 1989, Genome 31:104-111; Cooper et al., 1993,J. Biol. Chem. 268:11823-11829; Grilley et al., 1993, J. Biol. Chem.268:11830-11837). Strand cleavage enables one of three single-strandedexonucleases of E. coli (RecJ, Exol, ExoVII) to degrade thenon-methylated strand, which can then be re-synthesized by the E. coliPolIII holoenzyme complex (Lahue et al., 1989, Science 245:160-164). Thenet result is a strand-specific mismatch repair event.

[0006] Many genetic studies performed using E. coli support thisinterpretation. For example bacteria having a mutated mutH, mutL, ormutS gene exhibit a mutator phenotype that is presumed to be the resultof the increased probability of misincorporation errors leading tomutations (Demerec et al., 1957, Bact. Genet., Carnegie Inst. Wash.Yearbook 370:390-406; Miyake, 1960, Genetics 45:755-762; Siegel et al.,1967, J. Bacteriol. 94:38-47; Hill, 1970, Mutat. Res. 9:341-344).However, not all predictions arising from the E. coli Dam-instructedmodel agree with experimental results. For example, bacteria having amutation in each of the recj, exol, and exoVII genes do not exhibit amutator phenotype, suggesting that other exonuclease(s) or mechanism(s)are involved in the mismatch repair process.

[0007] Homologs of the procaryotic MutS and MutL proteins have beenidentified in eukaryotes (Fishel et al., 1993, Cell 75:1027-1038; Prollaet al., 1994, Science 265:1091-1093; Bronner et al., 1994, Nature368:258-261). MutH analogs appear to exist only in gram-negativebacteria.

[0008] Multiple MutS and MutL homologs have been identified in yeast andhuman cells which individually participate in such diverse activities asnuclear and organelle mismatch repair as well as distinct meioticfunctions (Fishel et al., 1997, Curr. Opin. Genet. Dev. 7: 105-113).Germ-line mutations of the human MutS and MutL Homologs, hMSH2, hMLH1,and hPMS2, have been found to be associated with the common cancerpredisposition syndrome, hereditary non-polyposis colorectal cancer(HNPCC; Bronner et al., 1994, Nature 368:258-261; Fishel et al., 1993,Cell 75:1027-1038). Yeast and human MutS and MutL homologs existprimarily as heterodimeric proteins. Yeast MSH2 protein has been foundto be associated with MSH3 or MSH6, and yeast MLH1 has been found to beassociated with PMS1. Human hMSH2 protein has been found to beassociated with hMSH3 or hMSH6 (also designated GTBP or p160 by someauthors), and human hMLH1 has been found to be associated with hPMS2 (Liet al., 1995, Proc. Natl. Acad., Sci. USA 92:1950-1954; Prolla et al.,1994, Science 265:1091-1093; Drummond et al., 1995, Science268:1909-1912; Marsischky et al., 1996, Gen. Dev. 10:407-420; Acharya etal., 1996, Proc. Natl. Acad. Sci. USA 93: 13629-13634). Furthermore,MSH2/MSH3 and MSH2/MSH6 protein complexes appear to possess overlappingand redundant mismatch binding activities (Acharya et al., 1996, Proc.Natl. Acad. Sci. USA 93:13629-13634; Risinger et al., 1996, NatureGenet. 14:102-105).

[0009] Classification of MutS and MutL homologs is based on the presencein the proteins of highly conserved regions of amino acid identity. Themost highly conserved region among MutS homologs includes approximately150 amino acids which comprise a helix-turn-helix domain associated witha Walker A adenine-nucleotide and magnesium binding motif (Walker etal., 1982, EMBO J. 1:945-951). This adenine nucleotide binding domainconstitutes more than 80% of the identifiable homology between MutShomologs (Fishel et al., 1997, Curr. Opin. Genet. Dev. 7:105-113). Bothpurified bacterial MutS homologs and purified yeast MutS homologspossess an intrinsic low-level ATPase activity (Haber et al., 1991,EMBO. J. 10:2707-2715; Chi et al., 1994, J. Biol. Chem. 269:29993-29997; Chi et al., 1994, J. Biol. Chem. 269:29984-29992; Alani etal., 1997, Mol. Cell Biol. 1 7: 2436-2447). This ATPase activity islikely to be important for the function of MutS homologs, as indicatedby the fact that mutation of conserved amino acid residues in theadenine nucleotide binding domain results in a dominant mutatorphenotype in both bacteria and yeast (Haber et al., 1991, EMBO. J.10:2707-2715; Wu et al., 1994, J. Bacteriol 176:5393-5400; Alani et al.,1997, Mol. Cell Biol. 1 7: 2436-2447). A central role for the adeninenucleotide binding domain is consistent with the ATP-dependenttranslocation model of mismatch repair proposed by Modrich andcolleagues (Allen et al., 1997, EMBO J. 16:4467-4476).

[0010] Genetic and biochemical studies of the human mismatch repairprocess indicate that it is similar to bacterial mismatch repair, exceptthat the physiologically relevant mechanism for directing strandspecificity is unknown (Miller et al., 1976, Proc. Natl. Acad. Sci. USA73:3073-3077; Glazer et al., 1987, Mol. Cell. Biol., 7:218-224; Holmeset al., 1990, Proc. Natl. Acad. Sci. USA 87:5837-5841; Thomas et al.,1991, J. Biol. Chem. 266:3744-3751; Fang et al., 1993, J. Biol. Chem.268:11838-11844; Longley et al., 1997, J. Biol. Chem. 272: 10917-10921).Purified hMSH2 protein binds mismatched nucleotides and DNA lesions(Fishel et al., 1994, Science 266:1403-1405; Fishel et al., 1994, CancerRes. 54:5539-5542; Mello et al., 1996, Chemistry & Biology 3:579-589),and the specificity and affinity of that recognition is enhanced byassociation of hMSH2 with hMSH3 or hMSH6 (Drummond et al., 1995; Acharyaet al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634; Palombo et al.,1996, Curr. Biol. 6:1181-1184).

[0011] Although the ability of MutS homologs to bind to mismatchedduplex DNA has been recognized (e.g. U.S. Pat. No. 5,556,750), methodsof using MutS homologs in vitro have been limited by a lack ofunderstanding regarding the properties of such homologs. A need remainsfor methods of binding MutS homologs and mismatched duplex DNA, whichmethods take advantages of the biochemical properties of such homologs.

[0012] Transgenic and Nullizygous Animals

[0013] The development of transgenic animals and nullizygous animalmodels has provided important new avenues for the study of specific genefunctions in differentiation, embryogenesis and neoplastic development(Palmiter et al., 1986, Ann. Rev. Genet. 20: 465-499). Transgenicanimals frequently serve as model systems for the study of variousdisease states and also provide an experimental system in which to testcompounds for their ability to regulate disease. Nullizygous animals aresimilarly useful as experimental systems for the testing of compoundsuseful for diagnosis, treatment, or both, of disease.

[0014] Lukkarinen et al. (1997, Stroke 28:639-645) teaches that geneconstructs which enable the generation of transgenic mice also enablethe generation of other transgenic rodents, including rats. Similarly,nullizygous mutations in a genetic locus of an animal of one species canbe replicated in an animal of another species having a genetic locushighly homologous to the first species. For example, many genetic lociare highly homologous among mammals, and even more highly homologousamong subgroups of mammals, such as among rodents.

[0015] The mutator hypothesis of tumorigenesis suggests that loss in anorganism of a chromosomal stability function, a chromosomal maintenancefunction, or both, results in an elevated mutation rate in the organism.An elevated mutation rate hastens accumulation of the numerous mutationsrequired for multistep carcinogenesis (Loeb, 1991, Cancer Res.51:3075-3079).

[0016] Loss of the function of p53 protein has been proposed to increasecellular hypermutability in an organism, thereby acceleratingtumorigenesis, although a clear role for p53 protein in genomicinstability remains controversial (Kastan et al., 1992, Cell 71:587-597;Fishel et al., 1997, Curr. Opin. Genet. Dev. 7:105-113). p53, the geneencoding p53 protein, is frequently mutated in a wide range of humancancers including, but not limited to, colonic tumors (Fearon et al.,1990, Cell 61:759-767). Transgenic mice nullizygous for p53 are viableand susceptible to tumorigenesis (de Wind et al., 1995, Cell 82:321-330;Reitmair et al., 1995, Nature Genet. 11:64-70; Donehower et al., 1992,Nature 356:215-221; Jacks et al., 1994, Curr. Biol. 4:1-7; Purdie etal., 1994, Oncogene 9:603-609).

[0017] Although nullizygous p53 mice can be used as models ofcarcinogenesis, the rates at which such mice develop tumors can beslower than what is desirable, particularly for large-scale screeningstudies involving numerous potential anti-cancer therapeutic orprophylactic compositions. What is needed is a transgenic mouse which,when exposed to a carcinogen, succumbs to tumorigenesis caused by thecarcinogen more readily than does a nullizygous p53 mouse and which,even when not exposed to an identifiable carcinogen, succumbs to tumorsmore readily than does a nullizygous p53 mouse.

[0018] Critical unmet needs also exist for animal models of programmedcell death (apoptosis) and of aging.

[0019] The present invention satisfies the needs identified above.

BRIEF SUMMARY OF THE INVENTION

[0020] The invention relates to a method of modifying a mismatchedduplex DNA. The method comprises contacting an MSH dimer and themismatched duplex DNA in the presence of a binding solution. In oneembodiment, the binding solution comprising a nucleotide selected fromthe group consisting of ADP and ATP, and the concentration of ATP in thebinding solution is less than about 3 micromolar The MSH dimer therebyassociates with the mismatched region of the mismatched duplex DNA, andthe mismatched duplex DNA is modified. In one embodiment, the MSH dimeris selected from the group consisting of a prokaryotic MSH homodimer, aprokaryotic MSH heterodimer, a eukaryotic MSH homodimer, and aeukaryotic MSH heterodimer. The MSH dimer may, for example, be ahomodimer of a MutS homolog selected from the group consisting of ahuman MutS homolog, a murine MutS homolog, a rat MutS homolog, aDrosophila MutS homolog, a yeast MutS homolog, and a Saccharomycescerevisiae MutS homolog. An example of a eukaryotic MSH homodimer is anMSH2 homodimer. The eukaryotic MSH heterodimer useful in this methodcomprises MutS homologs independently selected from the group consistingof an MSH2 protein, an MSH3 protein, an MSH4 protein, an MSH5 protein,and an MSH6 protein. By way of example, the MSH dimer may be selectedfrom the group consisting of an MSH2:MSH3 heterodimer, an MSH2:MSH6heterodimer, and an MSH4:MSH5 heterodimer. In another embodiment of thismethod, the prokaryotic MSH dimer is a homodimer of Escherichia coliMutS. Preferably, the MSH dimer is substantially purified.

[0021] According to this method, the concentration of ATP in the bindingsolution is preferably less than about 0.3 micromolar, or, morepreferably, the binding solution is substantially free of ATP. Inanother embodiment of this method, at least one of the MSH dimer and themismatched duplex DNA is bound to a support. In yet another embodiment,the mismatched duplex DNA has at least one free end. In still anotherembodiment, the mismatched duplex DNA comprises a DNA strand generatedby reverse transcription of mRNA obtained from an organism.

[0022] According to one aspect of this method, the mismatched duplex DNAcomprises a first DNA strand having a reference nucleotide sequence anda second DNA strand. The second strand may, for example, be selectedfrom the group consisting of a DNA strand obtained from an organism, aDNA strand obtained by amplification of at least a portion of apolynucleotide obtained from an organism, a DNA strand obtained bycleavage of a polynucleotide obtained from an organism, and a DNA strandobtained by reverse transcription of a polynucleotide obtained from anorganism. The second DNA strand may also comprise at least a portion ofa gene associated with a cancer in the organism. In one embodiment, theorganism is a human and the gene is selected from the group consistingof an oncogene and a tumor suppressor gene. By way of example, suchgenes include abl, akt2, apc, bcl2alpha, bcl2beta, bcl3, bcr, brcal,brca2, cb1, ccnd1, cdk4, crk-II, csf1r/fms, db1, dcc, dpc4/smad4, e-cad,e2f1/rbap, egfr/erbb-1, elk1, elk3, eph, erg, ets1, ets2, fer, fgr/src2,fli1/ergb2, fos, fps/fes, fral, fra2, fyn, hck, hek, her2/erbb-2/neu,her3/erbb-3, her4/erbb-4, hras 1, hst2, hstf1, ink4a, ink4b, int2/fgf3,jun, junb jund, kip2, kit, kras2a, kras2b, Ick, lyn, mas, max, mcc, met,mlh1, mos, msh2, msh3, msh6, myb, myba, mybb, myc, mycl1, mycn, nf1,nf2, nras, p53, pdgfb, pimi, pms1, pms2, ptc, pten, raf1, rbl, rel, ret,rosl, ski, srcl, tal1, tgfbr2, thral, thrb, tiaml, trk, vav, vhl, wafl,wntl, wnt2, wt1, and yes1. Preferably, the cancer is hereditarynon-polyposis colon cancer and the gene is selected from the groupconsisting of mlh1, msh2, msh3, msh6, pms1, and pms2. Alternately, thecancer may be selected from the group consisting of a leukemia, alymphoma, a meningioma, a mixed tumor of a salivary gland, an adenoma, acarcinoma, an adenocarcinoma, a sarcoma, a dysgerminoma, aretinoblastoma, a Wilms' tumor, a neuroblastoma, a melanoma, and amesothelioma.

[0023] In another aspect of this method, the mismatched duplex DNA andthe MSH dimer are contacted in the presence of at least onenon-mismatched duplex DNA. According to this aspect, the method mayfurther comprise separating the MSH dimer from the non-mismatched duplexDNA after contacting the mismatched duplex DNA and the MSH dimer. In oneembodiment, the method further comprising dissociating the mismatchedduplex DNA and the MSH dimer after separating the MSH dimer from thenon-mismatched duplex DNA and thereafter amplifying the mismatchedduplex DNA. The MSH dimer may be bound to a support prior to separatingthe non-mismatched duplex DNA from the MSH dimer and the non-mismatchedduplex DNA is separated from the MSH dimer in the presence of aseparating solution which is substantially free of ATP. In oneembodiment, this method further comprises releasing the mismatchedduplex DNA from the MSH dimer after separating the non-mismatched duplexDNA from the MSH dimer. If the mismatched duplex DNA has at least onefree end, it may be released from the MSH dimer by contacting the MSHdimer with a releasing solution. The releasing solution may, forexample, be selected from the group consisting of a solution comprisingATP and Mg²⁺ ions, a solution comprising ATP and a magnesium-chelatingagent, a solution comprising high salt, a solution comprising agamma-modified ATP analog and Mg²⁺ ions, and a solution comprising agamma-hydrolysis-resistant ATP analog and Mg²⁺ ions. Preferably, thereleasing solution comprises ATP and Mg ions. If the mismatched duplexDNA does not have a free end, it may be released from the MSH dimer bycontacting the MSH dimer with a releasing solution. This releasingsolution may be selected from the group consisting of a solutioncomprising a magnesium-chelating agent, a solution comprising high salt,a solution comprising a double-stranded DNA cleaving enzyme, ATP andMg²⁺ ions, a solution comprising a double-stranded DNA cleaving enzyme,a gamma-modified ATP analog, and Mg²+ions, and a solution comprising adouble-stranded DNA cleaving enzyme, a gamma-hydrolysis-resistant ATPanalog, and Mg²⁺ ions. According to one embodiment, after contacting themismatched DNA and the MSH dimer, the MSH dimer may be contacted with aMutL homolog.

[0024] In another aspect of this method, association of the MSH dimerwith the mismatched duplex DNA is detected after or while contacting theMSH dimer with the mismatched duplex DNA. Association of the MSH dimerwith the mismatched duplex DNA may be detected, for example, using anassay selected from the group consisting of a gel mobility shift assay,a filter binding assay, an immunological assay, a sedimentationcentrifugation assay, a spectroscopic assay, an optical affinity assay,a DNA footprint assay, and a nucleolytic cleavage protection assay.

[0025] In still another aspect of this method, the duplex DNA with whichthe MSH dimer is contacted does not have a free end. If the MSH dimer ispresent in molar excess with respect to the mismatched duplex DNA, thenan average of more than one the MSH dimer associates with one moleculeof the mismatched duplex DNA.

[0026] The invention also includes a method of modifying a mismatchedduplex DNA which does not have a free end. This method comprisingcontacting the mismatched duplex DNA and an MSH dimer having ADP boundthereto in the presence of a binding solution. The concentration of ATPin the binding solution is less than about 3 micromolar, and the homologassociates with the mismatched region of the mismatched duplex DNA,thereby modifying the mismatched duplex DNA.

[0027] The invention further includes a method of segregating amismatched duplex DNA from a population of DNA molecules. The methodcomprises contacting an MSH dimer and the population in the presence ofa binding solution and segregating the MSH dimer from the population.The binding solution comprises a nucleotide selected from the groupconsisting of ADP and ATP, an the concentration of ATP in the bindingsolution is less than about 3 micromolar. The MSH dimer associates withthe duplex DNA in the presence of the binding solution. When the MSHdimer is segregated from the population, the mismatched duplex DNA isalso segregated from the population.

[0028] The invention still further includes a method of detecting adifference between a sample nucleotide sequence and a referencenucleotide sequence. According to this method, a first DNA strand and asecond DNA strand are annealed to form a duplex DNA. The first DNAstrand has the sample nucleotide sequence, and the second DNA strand hasa nucleotide sequence which is complementary to the reference nucleotidesequence. If there is a difference between the sample nucleotidesequence and the reference nucleotide sequence, then the duplex DNA is amismatched duplex DNA. The duplex DNA and an MSH dimer are contacted inthe presence of a binding solution comprising a nucleotide selected fromthe group consisting of ADP and ATP. The concentration of ATP in thebinding solution is less than about 3 micromolar, and the MSH dimerassociates with the duplex DNA if the duplex DNA is a mismatched duplexDNA. According to this method, it is then determined whether the MSHdimer is associated with the duplex DNA molecule. Association of the MSHdimer with the duplex DNA molecule is an indication that there is adifference between the sample nucleotide sequence and the referencenucleotide sequence.

[0029] In addition, the invention includes a kit for separating amismatched duplex DNA from non-mismatched duplex DNAs. The kit comprisesat least two MutS homologs, a linker for binding the at least one of theMutS homologs to a support, and an additional reagent. The reagent may,for example, be selected from the group consisting of a nucleotide and areleasing solution, wherein the nucleotide is selected from the groupconsisting of ADP and ATP, and wherein the releasing solution comprisesMg²⁺ and a compound selected from the group consisting of ATP, agamma-modified ATP analog, and a gamma-hydrolysis-resistant ATP analog.

[0030] The invention also includes a method of determining whether amammal is predisposed for carcinogenesis. This method comprisesannealing a first DNA strand and a second DNA strand to form a duplexDNA. The first DNA strand has the nucleotide sequence of at least aportion of a gene selected from the group consisting of an oncogene anda tumor suppressor gene of the mammal. The second DNA strand has anucleotide sequence which is complementary to the consensus nucleotidesequence of this region. If there is a sequence difference between thefirst DNA strand and the second DNA strand then the duplex DNA is amismatched duplex DNA. The duplex DNA and an MSH dimer are contacted inthe presence of a binding solution comprising a nucleotide selected fromthe group consisting of ADP and ATP. The concentration of ATP in thebinding solution is less than about 3 micromolar, and the MSH dimerassociates with the duplex DNA if the duplex DNA is a mismatched duplexDNA. According to this method, it is determined whether the MSH dimer isassociated with the duplex DNA, whereby association of the MSH dimerwith the duplex DNA is an indication that the mammal is predisposed forcarcinogenesis.

[0031] The invention further includes a method of fractionating apopulation of duplex DNAs. This method comprises contacting thepopulation with an MSH dimer in the presence of a binding solutioncomprising a nucleotide selected from the group consisting of ADP andATP. The concentration of ATP in the binding solution is less than about3 micromolar, and the MSH dimer associates with at least one mismatchedduplex DNA in the population. The MSH dimer is segregated from thepopulation of duplex DNAs, whereby the mismatched duplex DNA is alsosegregated from the population. The population is thereby fractionated.

[0032] The invention still further includes a method of selectivelyamplifying at least one mismatched duplex DNA of a population of duplexDNAs. This method comprises contacting the population with an MSH dimerin the presence of a binding solution comprising a nucleotide selectedfrom the group consisting of ADP and ATP. The concentration of ATP inthe binding solution is less than about 3 micromolar, and the MSH dimerassociates with the mismatched duplex DNA. The MSH dimer is thereaftersegregated from the population of duplex DNAs, whereby the mismatchedduplex DNA is also segregated from the population of duplex DNAs. Themismatched duplex DNA is then amplified, whereby the mismatched duplexDNA is selectively amplified.

[0033] The invention also includes a method of determining whether thenucleotide sequence of a first copy of a genomic sequence differs fromthe nucleotide sequence of a second copy of the genomic sequence. Thismethod comprises amplifying a region of each of the first copy and thesecond copy of the genomic sequence to yield amplified first copies andamplified second copies. The amplified first copies and the amplifiedsecond copies are mixed and denatured to form a first mixture. Thenucleic acids in the first mixture are then annealed to form a secondmixture comprising duplex DNAs. If the nucleotide sequence of first copyand the nucleotide sequence of the second copy of the genomic sequencediffer, then at least some of the duplex DNAs in the second mixture aremismatched duplex DNAs. The annealed second mixture is contacted with anMSH dimer in the presence of a binding solution comprising a nucleotideselected from the group consisting of ADP and ATP. The concentration ofATP in the binding solution is preferably less than about 3 micromolar,whereby the MSH dimer associates with mismatched duplex DNA. Accordingto this method, it is then determined whether the MSH dimer isassociated with at least some of the duplex DNAs. Association of the MSHdimer with at least some of the duplex DNAs is an indication that thenucleotide sequence of the first copy of the genomic sequence differsfrom the nucleotide sequence of the second copy of the genomic sequence.

[0034] The invention further includes a composition for segregating amismatched duplex DNA from a population of duplex DNAs. The compositioncomprises an MSH heterodimer bound to a support.

[0035] The invention still further includes a kit for screening agenomic region for a nucleotide sequence which differs from a referencenucleotide sequence. This kit comprises a pair of primers complementaryto the ends of the region for amplifying the region, a DNA strand havingthe reference nucleotide sequence, and at least two MutS homologs.

[0036] The invention yet further relates to a nonhuman mammal which isnullizygous for both Msh2 and p53. The mammal does not express Msh2 orp53 and exhibits a phenotype selected from the group consisting ofinappropriate fetal apoptosis and a predisposition for carcinogenesis.

[0037] The invention also relates to a method of making a nonhumanmammal which is nullizygous for both Msh2 and p53, does not express Msh2or p53, and exhibits a phenotype selected from the group consisting of apredisposition for inappropriate fetal apoptosis and a predispositionfor carcinogenesis. This method comprises mating

[0038] a) a first parent mammal which comprises at least one null alleleof Msh2 and at least one null allele of p53 and

[0039] b) a second parent mammal comprising at least one null allele ofMsh2 and at least one null allele of p53. As a result of this mating, anon-human mammal is generated which is nullizygous for both Msh2 andp53, does not express Msh2 or p53, and exhibits a phenotype selectedfrom the group consisting of inappropriate fetal apoptosis and apredisposition for carcinogenesis.

[0040] The invention further relates to a method of determining whethera compound affects tumorigenesis in mammals. This method comprisesadministering the compound to a first nonhuman mammal which isnullizygous for both Msh2 and p53, does not express Msh2 or p53, andexhibits a predisposition for carcinogenesis. Tumor incidence in thefirst nonhuman mammal is compared with tumor incidence in a secondnonhuman mammal of the same type which is nullizygous for both Msh2 andp53, does not express Msh2 or p53, exhibits a predisposition forcarcinogenesis, and to which the compound is not administered. Adifference in tumor incidence in the first transgenic mammal comparedwith tumor incidence in the second transgenic mammal is an indicationthat the compound affects tumorigenesis in mammals.

[0041] The invention still further relates to a method of determiningwhether a compound affects a biological phenomenon in mammals. Thephenomenon may, for example, be selected from the group consisting ofapoptosis, aging, and fetal development. The method comprisesadministering the compound in utero to a first nonhuman mammalian embryowhich is nullizygous for both Msh2 and p53, does not express Msh2 orp53, and exhibits a predisposition for inappropriate fetal apoptosis.The development of the first nonhuman mammalian embryo is compared withthe development of a second nonhuman mammalian embryo of the same typewhich is nullizygous for both Msh2 and p53, does not express Msh2 orp53, exhibits a predisposition for inappropriate fetal apoptosis, and towhich the compound is not administered. A difference in the developmentof the first nonhuman mammalian embryo compared with the development ofthe second nonhuman mammalian embryo is an indication that the compoundaffects the biological phenomenon in mammals.

[0042] The invention yet further relates to a cell line which isnullizygous for both Msh2 and p53, does not express Msh2 or p53, andexhibits a phenotype selected from the group consisting of apredisposition for carcinogenesis and a predisposition for apoptosis.The cell line is made by culturing a cell obtained from the nonhumanmammal described herein.

[0043] The invention also relates to a method of determining whether acomposition affects expression of a gene selected from the groupconsisting of the p53 gene and a gene encoding a MutS homolog. Thismethod comprising administering the composition to a first non-humanmammal which is nullizygous for one of the p53 gene and the geneencoding a MutS homolog. A phenotype of the non-human mammal is comparedwith the phenotype of a second non-human mammal of the same type whichis not nullizygous for the one of the p53 gene and the gene encoding aMutS homolog, wherein the phenotype is selected from the groupconsisting of inappropriate fetal apoptosis and a predisposition forcarcinogenesis. A difference between the phenotype of the firstnon-human mammal and the phenotype of the second non-human mammal is anindication that the composition affects expression of the other of thep53 gene and the gene encoding a MutS homolog.

[0044] The invention further relates to a method of determining whethera composition affects expression of a gene selected from the groupconsisting of the p53 gene and a gene encoding a MutS homolog. Thismethod comprises administering the composition to a first cell derivedfrom a non-human mammal which is nullizygous for one of the p53 gene andthe gene encoding a MutS homolog. A phenotype of the first cell iscompared with the phenotype of a second cell derived from a non-humanmammal of the same type which is not nullizygous for the one of the p53gene and the gene encoding a MutS homolog, wherein the phenotype isselected from the group consisting of inappropriate fetal apoptosis anda predisposition for carcinogenesis. A difference between the phenotypeof the first cell and the phenotype of the second cell is an indicationthat the composition affects expression of the other of the p53 gene andthe gene encoding a MutS homolog.

[0045] The invention still further relates to a composition comprising ahuman MutS homolog fragment, wherein the fragment comprises a MutShomolog interaction region.

[0046] The invention yet further relates to a method of inhibitingassociation of a first human MutS homolog and a second human MutShomolog. This method comprises contacting at least one of the firsthuman MutS homolog and the second human MutS homolog with a human MutShomolog fragment comprising a MutS homolog interaction region.Inhibition of the first and the second human MutS homologs is thusinhibited.

[0047] The invention also relates to a composition comprisingsubstantially purified hMSH5.

[0048] The invention further relates to a composition comprising anisolated nucleic acid encoding hMSH5.

[0049] The invention still further includes an alternate method ofmodifying a mismatched duplex DNA. This method comprises contacting anMSH dimer and the mismatched duplex DNA in the presence of a bindingsolution comprising ADP. The concentration of ADP in the bindingsolution is at least about ten times the concentration of ATP, if ATP ispresent in the binding solution. The MSH dimer thereby associates withthe mismatched region of the mismatched duplex DNA and modifies themismatched duplex DNA.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0050]FIG. 1, comprising FIGS. 1A, 1B, 1C, 1D, 1E, and 1F, depictbinding of hMSH2:hMSH6 heterodimer to mismatched and non-mismatchedduplex DNA. FIG. 1A is an image of the results of a gel mobility shiftassay performed using the G/T-mismatched 81-base pair duplex DNAsubstrate described herein. The concentrations of heterodimer used inthe assay are indicated along the top of the image. The position of theS-shifted electrophoretic band is indicated by “S”. FIG. 1E is a graphwhich depicts the relationship between the concentration of heterodimerand the amount of product corresponding to the S-shifted electrophoreticband in FIG. 1A, as assessed using a phosphoimaging device. FIG. 1B isan image of the results of a gel mobility shift assay performed usingthe homologous 81-base pair duplex DNA substrate described herein. Theconcentrations of heterodimer used in the assay are indicated along thetop of the image. The position of the NS-shifted electrophoretic band isindicated by “NS”. FIG. 1F is a graph which depicts the relationshipbetween the concentration of heterodimer and the amount of productcorresponding to the NS-shifted electrophoretic band in FIG. 1B, asassessed using a phosphoimaging device. FIG. 1C is an image whichdepicts the results of a DNase footprint assay performed using the81-base pair G/T-mismatched duplex DNA substrate described herein. Theconcentrations of 81-base pair are indicated along the top of the image.The position of the G residue of the G/T-mismatched substrate isindicated by “G”, and the approximate region of the substrate protectedfrom DNase cleavage by the heterodimer is indicated by a vertical line.FIG. 1D is an image which depicts the results of a DNase footprint assayperformed using the homologous 81-base pair duplex DNA substratedescribed herein. The concentrations of heterodimer used in the assayare indicated along the top of the image. The position of the G/C basepair corresponding to the G/T-mismatched base pair of the mismatchedsubstrate is indicated by “^(G”).

[0051]FIG. 2, comprising FIGS. 2A, 2B, 2C, and 2D, depicts the resultsof gel mobility shift assays used to assess the ability of variousadenine nucleotides to dissociate MSH dimer from the mismatch site,corresponding to the S-shifted electrophoretic band, such that the MSHdimer, corresponding to the NS-shifted electrophoretic band, exhibitedDNA-associated diffusion. FIG. 2A is an image of an assay in which theproduct corresponding to the S-shifted electrophoretic band wasincubated in the presence of ATP at the concentration listed along thetop of the image. FIG. 2B is an image of an assay in which the productcorresponding to the S-shifted electrophoretic band was incubated in thepresence of adenosine-5′-O-3′-thiotriphosphate (ATP-gamma-S) at theconcentration listed along the top of the image. FIG. 2C is an image ofan assay in which the product corresponding to the S-shiftedelectrophoretic band was incubated in the presence of ADP at theconcentration listed along the top of the image. In FIGS. 2A, 2B, and2C, “-” indicates that no heterodimer was included in the assay mixture.FIG. 2D is a graph which depicts quantitated results obtained using theresults depicted in FIGS. 2A, 2B, and 2C, as assessed using aphosphoimaging device.

[0052]FIG. 3 is a bar graph which depicts the effect of selectednucleotides, deoxynucleotides, and nucleotide analogs on G/T mismatchbinding by the heterodimer, relative to the degree of binding observedin the absence of a (deoxy)nucleotide or analog. The effect of eachindicated (deoxy)nucleotide or analog was assessed at 25 micromolar(left bar of each pair) and at 250 micromolar (right bar of each pair).

[0053]FIG. 4, comprising FIGS. 4A and 4B depicts the effects of ATPhydrolysis or ADP binding by the hMSH2/hMSH6 heterodimer on mismatchedDNA binding. FIG. 4A is a graph depicting the results of gel mobilityshift assays performed in the presence or absence of 15 micromolar ATPand in the presence or absence of 15 micromolar ATP-gamma-S. Magnesiumchloride was added at the time designated “0”, and samples of the assaymixture were collected at the indicated times (in minutes). The bindingreaction in each mixture was halted by addition of 5 millimolar EDTA.FIG. 4B is a graph depicting the results of gel mobility shift assaysperformed in the presence of the indicated (in millimolar)concentrations of ATP or ADP or both.

[0054]FIG. 5 comprises FIGS. 5A and 5B. FIG. 5A is a graph which depictsthe results obtained in the assays described herein for detecting therate of a single round of ATP hydrolysis by the complex. FIG. 5B is agraph which depicts the results obtained in assays described herein fordetecting the rate of a single round of ATP hydrolysis by the complex inthe presence of selected amounts of mismatched DNA.

[0055]FIG. 6, comprising FIGS. 6A, 6B, 6C, and 6D, depicts the resultsof experiments performed to assess the effects of ATP, homologous DNA,or both, on the dissociation of the hMSH2:hMSH6 heterodimer from DNA.FIG. 6A is an image of the results obtained from gel mobility shiftassays in which heterodimer-bound mismatched DNA was incubated with ATPfor the time indicated in the image. FIG. 6B is an image of the resultsobtained from gel mobility shift assays in which heterodimer-boundmismatched DNA was incubated with ATP and a 400-fold excess ofhomologous DNA for the time indicated in the image. FIG. 6C is an imageof the results obtained from gel mobility shift assays in whichheterodimer-bound mismatched DNA was incubated with a 400-fold excess ofhomologous DNA for the time indicated in the image. FIG. 6D is an imageof the results obtained from gel mobility shift assays in which theheterodimer was incubated with homoduplex DNA probe for fifteen minutesat 37° C. (Lane A), the assay mixture was cooled to 4° C., and a1,100-fold excess of unlabeled competitor homoduplex DNA was added (LaneB). In each of FIG. 6A, 6B, 6C, and 6D, “-” indicates assay mixtureswhich did not comprise the heterodimer.

[0056]FIG. 7 is a diagram which depicts the model of the hMSH2:hMSH6heterodimer association with and dissociation from mismatched duplex DNAdescribed herein. The ADP-bound form of the heterodimer (“MSH₂”), whichis shown in the center of the diagram, is competent to bind mismatchedduplex DNA, as shown at the bottom of the diagram, but cannot diffusefrom the mismatch site on the DNA. Mismatched DNA-bound complex isenabled to diffuse to a different position on the DNA by displacement ofthe ADP molecule bound thereto by an ATP molecule (here indicated“*ATP”), which yields the ATP-bound form of the heterodimer. TheATP-bound form of the heterodimer is able to dissociate from a free endof the duplex DNA, but not from a blocked end of the duplex DNA. Afterdissociating from the duplex DNA, the ATP-bound form of the heterodimeris converted to the ADP-bound form by hydrolysis of theheterodimer-bound ATP molecule, catalyzed by intrinsic ATPase activityof the heterodimer.

[0057]FIG. 8 lists the nucleotide sequence of single nucleotide chainsof some of the 39-and 81-base pair DNA substrates described herein (SEQID NOS: 2, 3, 5, and 6).

[0058]FIG. 9, comprising FIGS. 9A, 9B, 9C, and 9D, is a series ofimages, each of which depicts a whole mount view of anMsh2^(−/−)p53^(−/−) embryo at day 11.5 of development. The embryodepicted in FIG. 9A is a male Msh2^(−/−)p53^(−/−) mouse embryo, andexhibits phenotypically normal embryonic development, relative to micehaving the same genotypic background. The embryos depicted in FIGS. 9B,9C, and 9D are female Msh2^(−/−)p53^(−/−) mouse embryos that arelittermates of the male mouse depicted in FIG. 9A. The female mouseembryos depicted in FIGS. 9B, 9C, and 9D exhibit developmental arresthaving a phenotype corresponding to that expected at day 9.5 ofembryonic development.

[0059]FIG. 10, comprising Panels A, B, C, D, E, and F, is a series ofimages, each of which depicts a paraffin embedded section obtained froman 11.5 day old female mouse embryo. The images in Panels A, C, and Eeach depict a section obtained from an 11.5 day old normal embryo. Theimages in Panels B, D, and F each depict a section obtained from an 11.5day old Msh2^(−/−)p53^(−/−) mouse embryo. The sections depicted inPanels A and B are at 100×magnification and are stained with hematoxylinand eosin. Magnification of the normal embryo is of the somite region ofa sagittal section. The sections depicted in Panels C and D are at 100×magnification and are chromogenically-TUNEL stained. The sectionsdepicted Panels E and F are at 40× magnification and arefluorescently-TUNEL stained. Cells undergoing apoptosis in normal femaleembryos were rare; chromogenically- and fluorescently-TUNEL stainedcells depicted in Panels C and E represent circumscribed apoptotic focinormally found in developing mouse embryos.

[0060]FIG. 11 is a graph which depicts Kaplan-Meier survivalprobabilities of Msh2^(−/−), p53^(−/−), and Msh2^(−/−)p53^(−/−) mice.

[0061]FIG. 12 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH3 polypeptides used to identify approximateboundaries of hMSH2-interaction regions of hMSH3. “Amino Acid Number”refers to the amino acid residues of hMSH3 which the correspondingIVTT-hMSH3 polypeptide comprised. The rectangular entities in thecentral part of the figure represent relative positions of the aminoacid residues which the corresponding IVTT-hMSH3 polypeptide comprisedwith respect to full length hMSH3, which is represented by polypeptide1). The symbol, Δ, indicates a deleted region of a polypeptide. Theshaded regions of polypeptide 1) represent the hMSH2-interaction regionsof hMSH3. “Interaction with hMSH2” indicates whether or not thecorresponding polypeptide interacted with GST-hMSH2.

[0062]FIG. 13 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH2 polypeptides used to identify approximateboundaries of hMSH3-interaction regions of hMSH2. “Amino Acid Number”refers to the amino acid residues of hMSH2 which the correspondingIVTT-hMSH2 polypeptide comprised. The rectangular entities in thecentral part of the figure represent relative positions of the aminoacid residues which the corresponding IVTT-hMSH2 polypeptide comprisedwith respect to full length hMSH2, which is represented by polypeptide1). The shaded regions of polypeptide 1) represent the hMSH3-interactionregions of hMSH2. “Interaction with hMSH3” indicates whether or not thecorresponding polypeptide interacted with GST-hMSH3.

[0063]FIG. 14 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH2 polypeptides used to identify the linearorientation of the hMSH3-interaction regions of hMSH2. “Amino AcidNumber” refers to the amino acid residues of hMSH2 which were present inthe corresponding IVTT-hMSH2 polypeptide. The rectangular entities inthe central part of the figure represent relative positions of the aminoacid residues which were present in the corresponding IVTT-hMSH2polypeptide with respect to full length hMSH3, which is represented bypolypeptide 1). The symbol, Δ, indicates a deleted region of apolypeptide. “Interaction with specific hMSH3 domains” indicates whetheror not the corresponding polypeptide interacted with a GST-hMSH3 fusionprotein comprising the amino-terminal (“NH₄ ⁺”) interaction region ofhMSH3 or with a GST-hMSH3 fusion protein comprising the carboxy-terminal(“COO⁻”) interaction region of hMSH3.

[0064]FIG. 15 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH6 polypeptides used to identify approximateboundaries of hMSH2-interaction regions of hMSH6. “Amino Acid Number”refers to the amino acid residues of hMSH6 which were present in thecorresponding IVTT-hMSH2 polypeptide. The rectangular entities in thecentral part of the figure represent relative positions of the aminoacid residues which were present in the corresponding IVTT-hMSH6polypeptide with respect to full length hMSH6, which is represented bypolypeptide 1). The shaded regions of polypeptide 1) represent thehMSH2-interaction regions of hMSH6. “Interaction with hMSH2” indicateswhether or not the corresponding polypeptide interacted with GST-hMSH2.

[0065]FIG. 16 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH2 polypeptides used to identify approximateboundaries of hMSH6-interaction regions of hMSH2. “Amino Acid Number”refers to the amino acid residues of hMSH2 which were present in thecorresponding IVTT-hMSH2 polypeptide. The rectangular entities in thecentral part of the figure represent relative positions of the aminoacid residues which the corresponding IVTT-hMSH2 polypeptide comprisedwith respect to full length hMSH2, which is represented by polypeptide1). The shaded regions of polypeptide 1) represent the hMSH6-interactionregions of hMSH2. “Interaction with hMSH3” indicates whether or not thecorresponding polypeptide interacted with GST-hMSH6.

[0066]FIG. 17 is a diagram which indicates the primary structure of³⁵S-labeled IVTT-hMSH2 polypeptides used to identify the linearorientation of the hMSH6-interaction regions of hMSH2. “Amino AcidNumber” refers to the amino acid residues of hMSH2 which were present inthe corresponding IVTT-hMSH2 polypeptide. The rectangular entities inthe central part of the figure represent relative positions of the aminoacid residues which were present in the corresponding IVTT-hMSH2polypeptide with respect to full length hMSH6, which is represented bypolypeptide 1). The symbol, Δ, indicates a deleted region of apolypeptide. “Interaction with specific hMSH6 domains” indicates whetheror not the corresponding polypeptide interacted with a GST-hMSH6 fusionprotein comprising the amino-terminal (“NH₄ ⁺”) interaction region ofhMSH6 or with a GST-hMSH6 fusion protein comprising the carboxy-terminal(“COO⁻”) interaction region of hMSH6.

[0067]FIG. 18 is a diagram which illustrates a model of hMSH2 consensusinteraction with hMSH3 or hMSH6. The interaction regions of hMSH2,hMSH3, and hMSH6 are indicated in gray and are connected with lines thatillustrate the specificity of each region to its correspondinginteraction partner region. The nucleotide binding regions of hMSH2,hMSH3, and hMSH6 are indicated as black boxes. The location ofHNPCC-associated mutations tested in these studies are illustrated asblack diamonds.

[0068]FIG. 19, comprising FIGS. 19A, 19B, and 19C, lists the nucleotidesequence of cDNA encoding hMSH5 (SEQ ID NO: 30) and the putative aminoacid sequence of hMSH5 (SEQ 1D NO: 29).

DETAILED DESCRIPTION OF THE INVENTION

[0069] The invention relates to a method of binding one or more MutShomolog (MSH) dimers to a mismatched duplex DNA. The invention alsorelates to methods of using adenine nucleotides to modulate recognitionof mismatched duplex DNA and to modulate DNA-associated diffusion of MSHdimers after binding of such dimers to mismatched duplex DNA. Theinvention further relates to a method of binding a complex comprising aMutL homolog and a MutS homolog to mismatched duplex DNA. The MutLhomolog interacts with the MutS homolog and influences the ability ofthe MutS homolog to bind with a mismatched region of the duplex DNA.

[0070] A Summary of Some of the Novel Properties of MutS Homologs andMutL Homologs

[0071] The compositions, kits, and methods of the invention may bebetter understood by understanding the novel properties of MutS homologsand MutL homologs which have been discovered by the inventors. Thissection presents merely a brief introduction to several theseproperties. It is understood that the operability of the compositions,kits, and methods of the invention does not depend upon the correctnessof the information provided in this section.

[0072] An important aspect of the invention is the discovery that MutShomolog (MSH) dimers and, in some organisms, MSH heterodimers, associatewith mismatched regions of a mismatched duplex DNA. Binding of a MutSdimer to mismatched DNA occurs when ADP, but not ATP, is bound to theMSH dimer. The MSH dimer may, for example, be in the form of an MSHhomodimer (e.g. an E. coli MutS dimer) or an MSH heterodimer (e.g. ahuman MSH heterodimer such as an hMSH2:hMSH3 dimer, an hMSH2:hMSH6dimer, or an hMSH4:hMSH5 dimer). This association may be effected eitherin vitro or in vivo.

[0073] ADP-bound MSH dimer associated with a mismatched region of amismatched duplex DNA does not move along the duplex DNA, but insteadremains located at the mismatched region. Exchange of ATP for the ADPbound to the MSH dimer confers to the MSH dimer DNA-associateddiffusibility, which means that the MSH dimer becomes able to move fromthe site of the mismatched region of the duplex DNA to another site onthe same duplex DNA. If the mismatched duplex DNA has a free end, thenthe DNA-associated diffusibility of an ATP-bound MSH dimer enables thedimer to the duplex DNA dissociate from the duplex DNA. If themismatched duplex DNA does not have a free end (e.g. the DNA is circularor has bulky moieties such as proteins bound to the ends thereof), thenneither the ADP-bound form or the ATP-bound form of the MSH dimer isable to dissociate from the duplex DNA.

[0074] Because MSH heterodimers, in their ATP-bound form, exhibitDNA-associated diffusibility with regard to the duplex DNA with whichthey are associated, an ATP-bound MSH dimer will not necessarily beassociated with the mismatched region of a mismatched duplex DNA, butinstead may have diffused away from the mismatched region tocomplementary region of the same mismatched duplex DNA. Thus, amismatched duplex DNA having one or more ATP-bound MSH dimers associatedtherewith is able to associate with another MSH dimer in an ADP-boundform. Therefore, numerous MSH dimers may be associated with a mismatchedduplex DNA by contacting the DNA with ADP-bound MSH dimers in thepresence of a binding solution which comprises ATP. It is understoodthat certain MSH homodimers (e.g. hMSH2 dimers; Fishel et al., 1994,Science 266:1403-1405) exhibit little or no alteration in activityassociated with adenine nucleotide binding, and may be useful for theseproperties. For example, hMSH2 binds a variety of mismatched nucleotidesbut remains unperturbed in the presence of either ADP or ATP (Fishel etal., 1994, Science 266:1403-1405).

[0075] MSH dimers exhibit an intrinsic ATP hydrolytic activity, and thishydrolytic activity is greatly enhanced in their non-DNA-associatedform, but not in their DNA-associated form. Thus, an ATP-bound MSH dimerassociated with DNA remains ATP-bound. However, ATP bound to an MSHdimer is rapidly converted to ADP if the dimer is not associated withDNA. Thus, the intrinsic ATPase activity exhibited by MSH dimerscatalyzes the transformation of an ATP-bound dimer (which cannotassociate with a mismatched region of DNA) to an ADP-bound dimer (whichcan associate with a mismatched region of DNA). I n addition, themismatched DNA-associated form of MSH dimers are able to more rapidlyexchange ATP in place of ADP bound to the dimer than MSH dimers notassociated with DNA or associated with non-mismatched DNA.

[0076] Without wishing to be bound by any particular theory ofoperation, binding of MSH dimers to mismatched duplex DNA may bevisualized as illustrated in FIG. 7. An ADP-bound MSH dimer associateswith the mismatched region of the DNA. Exchange of ATP in place of theADP bound to the MSH dimer enables the dimer to diffuse to a differentposition on the DNA. The DNA-associated ATP-bound MSH dimer cannotdissociate from a blocked end of the DNA in the presence of Mg²⁺, butcan dissociate from a free end of the DNA. Alternately, ATP-bound MSHdimer can be dissociated from DNA which does not have a free end in thepresence of EDTA or a high salt concentration. ATP-bound MSH dimer notassociated with DNA is able to hydrolyze the ATP moiety, yielding anADP-bound MSH dimer, which is then able to associate with a mismatchedregion of DNA.

[0077] An MSH dimer may be thought of as ‘molecular switch,’ wherein theADP-bound dimer represents an ‘ON’ state, and wherein the ATP-bounddimer represents an ‘OFF’ state. In the ‘ON’ state, the dimer is able toassociate with a mismatched region of DNA but is not able to diffuse toa different position on the DNA with which it is associated. In the‘OFF’ state, the dimer is not able to associate with a mismatched regionof DNA but is able to diffuse to a different position on the DNA withwhich it is associated. Recalling the involvement of MutS homologs inDNA mismatch repair and, as demonstrated herein, in control of the cellreplication cycle, it is understood that compounds which modulate thetransition of MSH dimers from the ‘ON’ to the ‘OFF’ state or vice versamay be used to modulate DNA mismatch repair, timing of and progressionthrough the cell replication cycle, and/or the physiological process(es)associated with either DNA mismatch repair or the cell replicationcycle.

[0078] A MutL homolog improve the intrinsic ATPase activity exhibited bya MSH dimer when the MutL homolog associates with the MSH dimer. MutLhomologs may thus be analogized to GTPase accelerating proteins(sometimes designated “GAP proteins”) which have been described in thecontext of G protein activity. Without wishing to be bound by anyparticular theory, it is thought that association of a MutL homolog witha MSH dimer increases the rate of dissociation of the ATP-bound MSHdimer from duplex DNA and increases the rate at which ATP is convertedto ADP by the non-duplex DNA-associated ATP-bound MSH dimer, therebyrendering the MSH dimer able to bind to a mismatched duplex DNA morerapidly than in the absence of the MutL homolog.

[0079] The biochemical properties of MutS homologs and MutL homologsdescribed in this section are used advantageously in the compositions,kits, and methods of the invention.

[0080] Definitions

[0081] As used herein, each of the following terms has the meaningassociated with it in this section.

[0082] A “MutS homolog” is a protein which comprises a region whichexhibits significant sequence similarity with at least one of thefollowing regions of the human MSH2 protein (wherein the regions areindicated by the numbers of the amino acid residues of MSH2 which,inclusively, bound the region; the corresponding amino acid sequences ofhMSH2 is indicated thereafter in parentheses):

[0083] Region I: hMSH2 amino acid residues 37-57 (LFDRGDFYTA HGEDALLAARE; SEQ ID NO: 24);

[0084] Region II: hMSH2 amino acid residues 336-368 (TPQGQRLVNQWIKQPLMDKN RIEERLNLVE AFV; SEQ ID NO: 25);

[0085] Region III: hMSH2 amino acid residues 635-662 (LKASRHACVEVQDEIAFIPN DVYFEKDK; SEQ ID NO: 26); Region I: hMSH2 amino acid residues37-57 (LFDRGDFYTA HGEDALLAAR E; SEQ ID NO:24); Region II: hMSH2 aminoacid residues 336-368 (TPQGQRLVNQ WIKQPLMDKN RIEERLNLVE AFV; SEQ IDNO:25); Region III: hMSH2 amino acid residues 635-662 (LKASRHACVEVQDEIAFIPN DVYFEKDK; SEQ ID NO:26); Region IV: hMSH2 amino acid residues667-770 (IITGPNMGGK STYIRQTGVI VLMAQIGCFV SEQ ID NO:27); and PCESAEVSIVDCILARVGAG DSQLKGVSTF MAEMLETASI LRSATKDSLI IIDELGRGTS TYDGFGLAWA ISEY;Region V: hMSH2 amino acid residues 812-852 (LTMLYQVKKG VCDQSFGIHVAELANFPKHV SEQ ID NO:28). IECAKQKALE L;

[0086] Region V: hMSH2 amino acid residues 812-852 (LTMLYQVKKGVCDQSFGIHV AELANFPKHV IECAKQKALE L; SEQ ID NO: 28).

[0087] The amino acid sequence of hMSH2 has been described (e.g. Fishelet al., 1993, Cell 75:1027). Preferably, the MutS homolog of theinvention comprises a region which exhibits significant sequencesimilarity with Region IV, and more preferably with both Region IV andRegion V. It is also preferred that the MutS homolog comprises aplurality of regions, each of which exhibits significant sequencesimilarity with one of Regions I-V of hMSH2, and more preferred that theMutS homolog comprises regions which independently exhibit significantsequence similarity with each of Regions I-V of hMSH2. Thus, MutShomologs which are included in the invention include, but are notlimited to Aquifex aeolicus MutS, Aquifex aeolicus MSH, Aquifiexpyrophilicus MutS, Arabidopsis thaliana MSH2, Arabidopsis thaliana MSH6,Azotobacter vinelandii MutS, Bacillus subtilis MutS, Bacillus subtilisMSH, Caenorhabdis elegans MSH4, Caenorhabdis elegans MSH5, Drosophilamelanogaster MSH2, Escherichia coli MutS, Homo sapiens MSH2, Homosapiens MSH3, Homo sapiens MSH4, Homo sapiens MSH5, Homo sapiens MSH6,Haemophilus influenzae type B MutS, Helicobacter pylori MSH, Musmusculus MSH2, Mus musculus MSH3, Mus musculus MSH6, Neurospora crassaMSH2, Rattus norvegicus MSH2, Saccharomyces cerevisiae MSH1,Saccharomyces cerevisiae MSH2, Saccharomyces cerevisiae MSH3,Saccharomyces cerevisiae MSH4, Saccharomyces cerevisiae MSH5,Saccharomyces cerevisiae MSH6, Saccharomyces pombe MSH1, Saccharomycespombe MSH2, Saccharomyces pombe Swi4, Saccharomyces pombe MutS,Salmonella typhimurium MutS, Synechocystis sp. MutS, Synechocystis sp.MSH, Thermus aquaticus MutS, Thermotoga maritima MutS, and Thermusthermophilus MutS, each of which proteins is described either herein orin the prior art.

[0088] A “MutL homolog” is a protein which exhibits significantsimilarity to the MutL protein of E. coli. MutL homologs include, butare not limited to, eukaryotic MLH1, MLH2, PMS 1, and PMS2 proteins.

[0089] A protein or a region of a protein exhibits “significantsimilarity” to another protein or a region of another protein if, whenthe two proteins or regions are compared in a selected alignment, atleast 50%, at least 70%, at least 85%, at least 95%, or at least 99% ofthe aligned amino acid residues of the two proteins or the two regionsare either identical or similar. Similar amino acid residues areindicated by the groups listed on the following lines:

[0090] glycine, alanine;

[0091] valine, isoleucine, leucine;

[0092] aspartic acid, glutamic acid;

[0093] asparagine, glutamine;

[0094] serine, threonine;

[0095] lysine, arginine; and

[0096] phenylalanine, tyrosine.

[0097] A “heterodimer” is a protein which comprises more than onesubunit, wherein at least one subunit has an amino acid sequences whichis different from the amino acid sequence of another subunit of the sameprotein. Heterodimers having an ‘A’ protein subunit and a ‘B’ proteinsubunit are herein designated “A:B heterodimers”.

[0098] A “DNA strand” is a single polydeoxyribonucleotide.

[0099] A “duplex DNA” is a molecule that comprises at least onepolydeoxyribonucleotide, wherein at least a portion of thepolydeoxyribonucleotide has a double-stranded, hydrogen bondedconformation.

[0100] A “mismatched” duplex DNA is a duplex DNA wherein at least oneDNA strand comprises a region which has at least one nucleotide residuethat is not base-paired with a complementary nucleotide residue andwhich is flanked by regions wherein at least about ten nucleotideresidues are all base-paired with complementary nucleotide residues.

[0101] A first region of an DNA “flanks” a second region of the DNA ifthe two regions are adjacent one another or if the two regions areseparated by no more than about 10 nucleotide residues, and preferablyno more than 1 nucleotide residue.

[0102] A “non-mismatched” duplex DNA is a duplex DNA wherein allnucleotide residues of the double-stranded portion thereof arebase-paired with complementary nucleotide residues.

[0103] “Complementary” refers to the broad concept of sequencecomplementarity between regions of two nucleic acid strands or betweentwo regions of the same nucleic acid strand. It is known that an adenineresidue of a first nucleic acid region is capable of forming specifichydrogen bonds (“base pairing”) with a residue of a second nucleic acidregion which is antiparallel to the first region if the residue isthymine or uracil. Similarly, it is known that a cytosine residue of afirst nucleic acid strand is capable of base pairing with a residue of asecond nucleic acid strand which is antiparallel to the first strand ifthe residue is guanine. A first region of a nucleic acid iscomplementary to a second region of the same or a different inucleicacid if, when the two regions are arranged in an antiparallel fashion,at least one inucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

[0104] A chemical entity such as a molecule is “bound” to anotherchemical entity if at least one portion of each of the two chemicalentities are covalently or non-covalently bonded to one another in anessentially fixed position. By way of example, as described herein, anADP-bound form of an MSH dimer is bound to a mismatched region of aduplex DNA because the MSH dimer predominantly associates with theduplex DNA at the location of the mismatch.

[0105] A chemical entity such as a molecule is “associated” with anotherchemical entity if at least one of the chemical entities can change itsposition relative to the other without becoming dissociated therefrom.By way of example, as described herein, an ATP-bound form of an MSHdimer is associated with a mismatched duplex DNA because the MSH dimercan diffuse to a different position on the DNA without dissociatingtherefrom.

[0106] A duplex DNA is “modified” if a chemical entity such as amolecule is bound to, associated with, or dissociated from the duplexDNA, or if the duplex DNA is segregated from a population of DNAmolecules.

[0107] A duplex DNA has a “free end” if the duplex DNA is not circularand if both ends of the duplex DNA are not blocked.

[0108] An end of a duplex DNA is “blocked” if a bulky moiety is bound toa portion of the duplex DNA between a reference point on the duplex DNAand the end of the duplex DNA.

[0109] A “bulky moiety” bound to a portion of a duplex DNA is anychemical entity which has a size sufficient to prevent sliding of anATP-bound MSH dimer along the DNA duplex from a location on one side ofthe bulky moiety to a location on the other side of the bulky moiety.Examples of bulky moieties include proteins, metallic, glass, orpolymeric surfaces, and the like.

[0110] A “gamma-modified ATP analog” is an ATP molecule which has an agroup attached to the gamma phosphodiester moiety thereof, whereby thebeta-gamma phosphodiester linkage is cleaved by an MSH dimer with anefficiency less than 25% of the efficiency with which ATP is hydrolyzedby the MSH dimer. By way of example, ATP-gamma-S is a gamma-modified ATPanalog.

[0111] A “gamma-hydrolysis-resistant ATP analog” is an ATP moleculewhich has an altered beta-gamma phosphodiester linkage chemistry wherebythe altered beta-gamma phosphodiester linkage cannot be cleaved beeither the intrinsic ATP hydrolytic activity of an MSH dimer or by theATP hydrolytic activity of an MSH dimer-MutL homolog complex. Examplesof gamma-hydrolysis-resistant ATP analogs include, but are not limitedto ATP-PNP and ATP-PCP, which are compounds well known and described inthe art.

[0112] A solution is “substantially free” of ATP when the concentrationof ATP is very low (e.g. less than 30 nanomolar, and preferably lessthan 1 nanomolar).

[0113] The term “substantially pure” describes a compound, e.g., aprotein or polypeptide which has been separated from components whichnaturally accompany it. Typically, a compound is substantially pure whenat least 10%, more preferably at least 20%, more preferably at least50%, more preferably at least 60%, more preferably at least 75%, morepreferably at least 90%, and most preferably at least 99% of the totalmaterial (by volume, by wet or dry weight, or by mole percent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method, e.g., in the case of polypeptides bycolumn chromatography, gel electrophoresis or HPLC analysis. A compound,e.g., a protein, is also substantially purified when it is essentiallyfree of naturally associated components or when it is separated from thenative contaminants which accompany it in its natural state.

[0114] “Nullizygous” refers to an animal which possesses a pair of nullmutant alleles at a given genetic locus. Hence, a nullizygous Xxx mouse(wherein Xxx is any gene normally present in a mouse) does not possess afunctional Xxx gene, whereas a wild-type mouse may possess one or twofunctional copies of the Xxx gene. To illustrate the notation usedherein, the term “nullizygous Xxx mouse” is synonymous with the term“Xxx^(−/−) mouse.” Similarly, a “heterozygous Xxx mouse” has onefunctional Xxx allele and one non-functional Xxx allele, and issynonymous with the term “Xxx^(+/+) mouse.” A “wild type mouse” has atleast one copy, and possibly two copies, of a functional Xxx allele, andis synonymous with the term “Xxx^(+/±) mouse.” A “homologous wild typemouse” has two copies of a functional Xxx allele, and is synonymous withthe term “Xxx^(+/+) mouse.”

[0115] As used herein, an “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositions andmethods of the invention for associating a MSH dimer with a mismatchedduplex DNA. The instructional material of the kit of the invention may,for example, be affixed to a container which contains the dimer or beshipped together with a container which contains the dimer.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thedimer be used cooperatively by the recipient.

[0116] A solution comprises “high salt” if the concentration of one ormore salts in the solution is, cumulatively, at least about 1 molar,preferably at least about 3 molar.

[0117] A “double-stranded DNA-cleaving enzyme” is an enzyme whichcatalyzes hydrolysis of both strands of a duplex DNA, leaving eitherblunt or staggered ends. Examples of double-stranded DNA-cleavingenzymes include, but are not limited to, restriction endonucleases.

[0118] Description

[0119] The invention relates to a method of modifying a mismatchedduplex DNA. The method comprises contacting a MutS homolog (MSH) dimerand the mismatched duplex DNA in the presence of a binding solution. Thebinding solution comprises either ADP and ATP, and the concentration ofATP in the binding solution is less than about 3 micromolar, preferablyless than about 0.3 micromolar, and more preferably wherein the bindingsolution is substantially free of ATP. Alternately, ADP is used in theabsence of ATP, or at least in excess with respect to ATP (i.e. ADP at a2-fold, 10-fold, or 100-fold or greater excess relative to ATP). The MSHdimer thereby binds ADP. When the ADP-bound MSH dimer is contacted withthe mismatched duplex DNA, the dimer associates with the mismatchedregion of the DNA, thus forming a modified mismatched duplex DNA.

[0120] The MSH dimer may be a homodimer or a heterodimer of any MutShomolog which is presently known to comprise or is discovered tocomprise one or more which exhibits significant sequence similarity withat least one of Region I-V of human MSH2 (hMSH2), as described herein.The MutS homolog may be a prokaryotic MutS homolog or a eukaryotic MutShomolog. Preferably, the MutS homolog is a heterodimer, more preferablya heterodimer comprising MutS homologs obtained from a single species oforganism. Thus, by way of example, the MSH dimer useful in the methods,kits, and compositions of the invention may be the E. coli MutS protein,an hMSH2 homodimer, a heterodimer comprising hMSH2 and either hMSH3 orhMSH6, a heterodimer comprising hMSH4 and hMSH5, a yeast MSH2 proteinhomodimer, a heterodimer comprising yeast MSH2 and either yeast MSH3 oryeast MSH6, a homodimer of a rat MSH2 (e.g. GenBank accession numberX93591), a dimer of a Xenopus homolog of hMSH2 (Varlet et al., 1994,Nucl. Acids Res. 22:5723-5728), a homodimer of Drosophila MSH2 (e.g.GenBank accession number U17893), a homodimer of murine MSH2 (e.g.GenBank accession number X93591, Varlet et al., 1994, Nucl. Acids Res.22:5723-5728), a heterodimer comprising murine MSH2 and either murineMSH3 (e.g. Rep-3; Linton et al., 1989, Mol. Cell. Biol. 9:3058-3072;Smith et al., 1990, Mol. Cell. Biol. 10:6003-6012) or murine MSH6 (e.g.Gen Bank accession number U42190), and the like. The MutS homolog of theMSH dimer used in the compositions, kits, and methods of the inventionmay also be any of the 41 MutS homologs and presently listed in the NCBIdatabase. It is understood that, given the high degree of similarityamong mammalian MutS homologs (Fishel et al., 1997, Curr. Op. Genet.Develop. 7:105-113), a dimer of any mammalian hMSH2 homolog can be usedin the methods of the invention.

[0121] The mismatched duplex DNA molecule useful in the methods of theinvention may be any duplex DNA molecule having at least one mismatchedregion. By way of example, the DNA molecule may be a linear DNAmolecule, a circularized DNA molecule such as a plasmid or a viralgenome, a chromosome, a cDNA generated by reverse transcription of anRNA molecule, a PCR primer, a PCR product, a complex formed between asingle-stranded DNA probe and another single-stranded DNA molecule, andthe like. The mismatched region may be any region of a duplex DNAmolecule in which the two DNA strands of the molecule are not completelycomplementary. By way of example, the mismatched region may comprise oneor more pairs of mismatched nucleotides in an otherwise complementaryregion of a duplex DNA molecule, a region of a duplex DNA moleculewherein a thymine dimer exists on one DNA strand of the molecule, aregion of a duplex DNA molecule comprising a nucleotide which has beencovalently modified by an agent capable of reacting with a nucleotide,such as cisplatin, a region of a duplex DNA molecule which comprises analkyl-O-6-methyl guanine residue, a region of a duplex DNA moleculewhich comprises a single stranded loop of one or more nucleotides, aregion of a duplex DNA molecule which comprises a pyrimidine dimer, andthe like.

[0122] While any amount of ADP can be used in the binding solution ofthe method of the invention, it is preferred that the homolog becontacted with the mismatched duplex DNA in the presence of a bindingsolution comprising at least about 100 nanomolar ADP, preferably atleast about 6 micromolar ADP, and more preferably at least about 60micromolar ADP. As described with greater particularity in Example 1,ATP displaces ADP from the MSH dimer when the dimer is associated with amismatched region of duplex DNA. Thus, it is important either that theconcentration of ATP in the solution be minimized, for example bymaintaining the concentration of ATP lower than about 3 micromolar,preferably lower than about 0.3 micromolar, and more preferably lowerthan about 10 nanomolar, or that the ratio of the concentration of ADPin the solution to the concentration of ATP in the solution be greaterthan a minimum value, such as about two, and preferably greater thanabout eight, and even more preferably greater than about sixteen.Preferably, the solution is substantially free of ATP or the ratio ofADP to ATP is much greater than sixteen (e.g. [ADP]:[ATP] is 100:1 orgreater).

[0123] It is understood that gamma-hydrolysis-resistant ATP analogs,certain other ATP analogs, and other ADP analogs may be bound to an MSHdimer, and that these analog-bound dimers will associated withmismatched duplex DNA. By way of example, MSH2:MSH6 dimers willassociate with mismatched DNA in the presence of either ATP-PNP orATP-PCP.

[0124] The MSH dimer that is useful in the compositions, kits, andmethods of the invention may be used in a variety of states of purity orisolation. For example, the dimer may be present in a liquid which avariety of other proteins, nucleic acids, lipids, single strandednucleic acids, non-mismatched duplex DNA, and the like, it beingunderstood that if the dimer is used in the form of a mismatched duplexDNA-containing liquid then it may be necessary to dissociate, andpossibly to separate, the dimer from the mismatched DNA prior to usingit in the compositions, kits, and methods of the invention. Preferably,the MSH dimer is substantially purified.

[0125] In many of the compositions, kits, and methods of the invention,the MSH dimer or the mismatched duplex DNA may bound to a support.Furthermore, each of the MSH dimer and the mismatched duplex DNA may bebound to different supports.

[0126] The MSH dimer or a MutS homolog of the dimer may be bound to asupport using any known method for attaching a protein to a surface. Forexample the MutS homolog may be bound to a support by way of an antibodywhich is covalently bound to the support and which has a variable regionwhich specifically binds to the MutS homolog. By way of example, anantibody which specifically binds to hMSH2 such as the antibodydescribed by Kinzler et al. (PCT publication number WO96/41192) may beused to bind an hMSH2 protein dimer or a complex comprising an hMSH2protein molecule and either an hMSH3 protein molecule or an hMSH6protein molecule to a support to which the antibody is fixed. Methods offixing an antibody to a support have been described in the art (e.g.Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y.). Alternately, covalent, ionic, hydrophobic, or other typesof bonding forces may be used to attach an MSH dimer or a MutS homologto a support.

[0127] The duplex DNA molecule may be bound to a support using any knownmethod for attaching a nucleic acid to a support. By way of example, thenucleic acid may be covalently linked to a biotin molecule and thesupport may be linked to or coated with a streptavidin molecule, wherebythe streptavidin molecule is capable of binding the biotin molecule,thereby linking the nucleic acid to the support. Further by way ofexample, the duplex DNA may be covalently attached to a chemicalsubstituent present on a surface of the support. Alternately, covalent,ionic, hydrophobic, or other types of bonding forces may be used toattach the duplex DNA to the support.

[0128] Supports to which an MSH dimer, a MutS homolog, or a duplex DNAmolecule may be bound include any support known in the art for use in invitro or in vitro biochemical or medical applications. By way ofexample, and not limitation, such supports include latex and otherpolymeric beads, particles, plates, supports, chromatography media,implants, drug delivery vehicles, metal and glass surfaces, gelatinoussurfaces such as agarose, alginates, and polyacrylamides, and the like.It is important that the ability of the MutS homolog monomers or MSHdimers which are bound to the support be attached in such a way that theability of the monomers to dimers to attain altered conformations is notsignificantly hindered. It is understood that, for example, by isolatingantibodies which specifically bind to various epitopes on the monomer ordimer surface, a variety of antibodies may be isolated an used to bindmonomers or dimers to a support. By assaying the ability of thesupport-bound monomers or dimers to bind to mismatched DNA in thepresence of ADP, for example as described herein, an antibody or othersupport which attaches the monomers or dimers to a support withouthindering their ability to bind mismatched duplex DNA may be identified.Such methods are routine in the art of protein immobilization and arenot further described herein.

[0129] As disclosed herein, after an ADP-bound MSH dimer binds to amismatched region of a duplex DNA, exchange of ATP for the ADP bound tothe dimer results in release of the dimer from the mismatched region,whereby the ATP-bound dimer is enabled to diffuse to a differentposition on the DNA. If the dimer is able to diffuse to a free end ofthe duplex DNA, the dimer may dissociate from the duplex DNA.

[0130] If the duplex DNA does not have a free end, then ATP-bound dimermay diffuse away from the mismatched region of the duplex DNA, but maynot dissociate from the DNA. Thus, if the duplex DNA does not have afree end, a plurality of copies of the dimer may be associated with theDNA in the presence of ATP. No upper limit is known for the number ofdimers which may be associated with the DNA, but it is contemplated thatthis number is roughly proportional to the length of the duplex DNA. Itis understood that association of multiple copies of an MSH dimer with amismatched duplex DNA may be advantageous in situations in whichassociation of the dimer with the DNA is to be detected. Multiple copiesof the dimer may boost the detection limit of the DNA to be detected,increasing the signal-to-noise ratio of the detection method.

[0131] Duplex DNA not having a free end may be circular DNA or it may belinear DNA wherein both ends of the DNA are blocked. Ends of duplex DNAmay be blocked by binding bulky moieties such as proteins to the DNAeither directly (e.g. by covalently attaching the protein to the DNA orby binding the protein to the DNA non-covalently with high affinity) orvia a linker (e.g. by biotinylating the DNA and binding an avidin suchas streptavidin to the biotin moiety). Bulky moieties which may be usedto block the ends of duplex DNA include, but are not limited to,proteins, supports, hairpin DNA structures, stem-and-loop DNAstructures, and multiple a stem-and-loop DNA structures. Association ofMSH dimers with DNA having one, two, or no free ends is expresslycontemplated.

[0132] The mismatched duplex DNA to which an MSH dimer is to be boundmay, for example, comprise a first DNA strand having a referencenucleotide sequence and a second DNA strand selected from the groupconsisting of a DNA strand obtained from an organism, a DNA strandobtained by amplification of at least a portion of a polynucleotideobtained from an organism, a DNA strand obtained by cleavage of apolynucleotide obtained from an organism, and a DNA strand obtained byreverse transcription of a polynucleotide obtained from an organism. Byway of example, the second DNA strand may comprise at least a portion ofa gene associated with a cancer in the organism. This gene may, forexample, be any of a number of oncogenes and tumor suppressor geneswhich are known in the art. Examples of such genes include, for example,abl, akt2, apc, bcl2alpha, bcl2beta, bcl3, ber, brcal, brca2, cb1,ccndl, cdk4, crk-II, csflr/fms, db1, dcc, dpc4/smad4, e-cad, e2fl/rbap,egfr/erbb-1, elk1, elk3, eph, erg, ets1, ets2, fer, fgr/src2,flil/ergb2, fos, fps/fes, fral, fra2, fyn, hck, hek, her2/erbb-2/neu,her3/erbb-3, her4/erbb-4, hras1, hst2, hstfl, ink4a, ink4b, int2/fgf3,jun, junb, jund, kip2, kit, kras2a, kras2b, Ick, lyn, mas, max, mcc,met, mlh1, mos, msh2, msh3, msh6, myb, myba, mybb, myc, mycl1, mycn,nf1, nf2, nras, p53, pdgfb, pim1, pms1, pms2, ptc, pten, raf1, rb1, rel,ret, ros1, ski, src1, tal1, tgfbr2, thra1, thrb, tiam1, trk, vav, vh1,waf1, wnt1, wnt2, wt1, and yes1. These genes are described in variouspublicly available databases, including the U.S. National CancerInstitute/National Center for Biotechnology Information Cancer GenomeAnatomy Project database. Various accession numbers for these genes arelisted in Table 1. TABLE 1 Entrez PubMed UniGene Gene Symbol AccessionUID CID CGAP ABL X16416 90082420 Hs.82576 AA601510 AKT2 M95936 93028445Hs.37433 AA505663 APC M74088 91335210 Hs.75081 AA592971 BCL2ALPHA M1399486259760 Hs.89534 AA577385 BCL2BETA M13995 86259760 Hs.99916 BCL3 M3173290199880 Hs.31210 AA527996 BCR Y00661 85240564 Hs.2557  AA592930 BRCA1U14680 95025896 Hs.66746 AA484941 BRCA2 X95161 96112016 Hs.34012AA215820 CBL X57110 92228506 Hs.99980 CCND1 M64349 91235304 Hs.82932AA592929 CDK4 U37022  8528263 Hs.95577 AA483705 CRK-II D10656 92334347Hs.16   CSF1R/FMS X03663 86175013 Hs.75116 AA595091 DBL X12556 89052660Hs.89543 DCC X76132 95011532 Hs.68149 DPC4/SMAD4 U44378 96144684Hs.75862 AA576881 E-CAD Z13009 93211394 Hs.82004 AA603448 E2F1/RBAPM96577 92346720 Hs.89494 EGFR/ERBB-1 X00588 84219729 Hs.77432 AA587386ELK1 M25269 89203250 Hs.1399  AA576028 ELK3 Z36715 95047310 AA262193 EPHM18391 88070650 Hs.1113  ERG M17254 87263429 Hs.70388 ETS1 X1479889083219 ETS2 J04102 89042086 Hs.85146 AA480196 FER J03358 89261786AA534773 FGR(SRC2) M12502 85205090 Hs.1422  FLI1/ERGB2 M98833 93075640Hs.736  FOS V01512 83221560 Hs.25647 AA514238 FPS/FES X06292 86055727Hs.7636  FRA1 X16707 90191709 Hs.4245  FRA2 X16706 90191709 Hs.89765AA601534 FYN M14333 86287278 Hs.75390 AA524156 HCK M16591 87257942Hs.77058 HEK M83941 92179233 HER2/ERBB-2/ X03363 86118663 Hs.46254AA508596 NEU HER3/ERBB-3 M29366 90083234 Hs.82186 AA570304 HER4/ERBB-4L07868 93189574 Hs.1939  HRAS1 V00574 83141783 Hs.37003 AA483837 HST2X63454 92195660 HSTF1 J02986 87204251 Hs.1755  INK4A L27211 94081956Hs.1174  AA557137 INK4B L36844 94359613 INT2/FGF3 X14445 89239468Hs.37092 AA525331 JUN J04111 89057892 Hs.78465 AA582267 JUNB M2903990090625 Hs.89792 AA503220 JUND X56681 91232849 Hs.2780  AA533575 KIP2D64137 96209909 Hs.9039  AA524076 KIT X06182 88111521 Hs.81665 AA552932KRAS2A L00045 83271513 KRAS2B X01669 85087906 LCK X13529 89123626Hs.1765  AA282059 LYN M16038 87172710 Hs.80887 AA524487 MAS M1315086218084 Hs.99900 MAX M64240 91173288 Hs.89500 AA592936 MCC M6239791164855 Hs.1345  MET J02958 87317655 Hs.35379 MLH1 U07343  8145827Hs.57301 MOS J00119 82275068 MSH2 U04045 94084796 Hs.78934 AA502616 MYBM15024 87092302 Hs.1334  AA535078 MYBA X66087 Hs.2537  AA459003 MYBBX13293 89083548 Hs.74605 AA603093 MYC X00196 84131953 Hs.79070 MYCL1M19720 88094386 Hs.92137 MYCN Y00664 88202932 Hs.25960 AA548970 NF1M89914 90335969 Hs.37170 AA534609 NF2 L11353 93201601 Hs.902  AA617825NRAS X02751 85269641 Hs.82602 AA558915 P53 K03199 85267676 Hs.1846 AA514357 PDGFB M12783 87217119 Hs.1976  PIM1 M27903 90382681 Hs.81170AA251525 PTC U59464  8658145 Hs.54503 RAF1 X03484 86120351 Hs.85181AA578685 RB1 M15400 87149066 Hs.75770 AA594282 REL X75042 89330980Hs.44313 AA279536 RET M16029 87257826 Hs.6253  ROS1 M34353 90280463Hs.1041  SKI X15218 89345144 Hs.2969  AA258011 SRC1 M16243 87257903Hs.65442 AA523427 TAL1 M29038 90099309 Hs.73828 AA551582 TGFBR2 M8507992154690 Hs.82028 AA515322 THRA1 Y00479 88067793 Hs.724  AA602782 THRBX04707 87090375 AA577807 TIAM1 X86351 96129318 Hs.3205  TRK M2310289181575 Hs.85844 VAV X16316 90005432 VHL L15409 93262488 Hs.78160 WAF1L25610 94061996 Hs.74984 AA614342 WNT1 X03072 86055728 WNT2 X0787689005063 Hs.89791 AA601910 WT1 X51630 90158822 Hs.1145  YES1 M1599087172733 Hs.75680 AA502695

[0133] In a preferred embodiment, the gene associated with a cancer is agene associated with hereditary non-polyposis colon cancer. For example,the gene may be selected from the group consisting of mlh1, msh2, msh2,msh3, msh6, pms1, and pms2. In another embodiment the gene may be a geneassociated with a cancer selected from the group consisting of aleukemia, a lymphoma, a meningioma, a mix tumor of a salivary gland, andadenoma, a carcinoma, and adenocarcinoma, a sarcoma, a dysgerminoma, aretinoblastoma, a Wilms' tumor, a neuroblastoma, a melanoma, and amesothelioma.

[0134] If an MSH dimer is contacted with a mixture of mismatched duplexDNA and non-mismatched duplex DNA, the dimer will preferentiallyassociate with the mismateched duplex DNA. The mis-matched duplex DNA isthere by labled differently than the non-mismatched duplex DNA, and MSHdimer associated with mismatched duplex DNA may be detected as describeherein or separated from the non-mismatched DNA. By separating the dimerfrom non-mismatched duplex DNA, the mismatched duplex DNA bound to thedimer is separated from the non-mismatched duplex DNA. Furthermore,mismatched duplex DNA may be dissasociated from the dimer afterseparating it from the non-mismatched duplex DNA.

[0135] Methods of detecting an MSH dimer associated with mismatchedduplex DNA include, but are not limited to, electrophoretic gel mobilityshift assays, HPLC and other column and thin layer chromatographicmethods, filter binding assays, immunologic detection methods such asELISA, tagged antibody, and precipitation assays, centrifugalsedimentation methods, optical affinity sensing, ‘footprint’ and othernucleolytic cleavage protection assays, and spectroscopic assays.

[0136] In a preferred method of detecting specific binding of the MutShomolog to the duplex DNA molecule, an optical affinity biosensor system(OABS) is used to detect specific binding. In an OABS system such as theIASYSTM system (Affinity Sensors, Cambridge, United Kingdom), bindingand dissociation events can be detected as one molecule in solutionbinds to or dissociates from another molecule immobilized on a detectorsurface of the system. Thus, an OABS may be used to detect specificbinding between an MSH dimer and a mismatched duplex DNA in any of themethods of the invention by immobilizing either the MSH dimer or themismatched duplex DNA on the detector surface of the OABS. Specificbinding may be differentiated from non-specific binding by comparingbinding of an MSH dimer to a duplex DNA molecule known to comprise amismatched region and binding of the homolog to a duplex DNA moleculeknown not to comprise a mismatched region.

[0137] By way of example, the separation of a mismatched duplex DNA froma population of duplex DNAs may be achieved by binding an MSH dimer to asupport, contacting the support with the population of duplex DNAs, andrinsing the support with a separating solution which does not comprisethe population of duplex DNAs. If the mismatched duplex DNA has a freeend, then the separating solution is preferably substantially free ofATP. In this example, a mismatched duplex DNA in the population ofduplex DNAs binds to the MSH dimer and thereby becomes associated withthe support. The mismatched duplex DNA is segregated from the otherduplex DNAs of the population by rinsing the support with the separatingsolution, which carries the non-mismatched DNA molecules away from thesupport. Thus, according to this example, the mismatched duplex DNA isphysically separated from the non-mismatched duplex DNAs of thepopulation.

[0138] It is not necessary that the just-described method result inseparation of the mismatched duplex DNA from the population such thatthe molecule and the population are contained in different containers atthe conclusion of the method. By way of example, it is sufficient in theOABS described herein that a mismatched duplex DNA comprising a regionassociate with the detector surface of the OABS and that non-mismatchedduplex DNAs do not associate with the detector surface of the OABS.Thus, for example, in OABS methods for detection of mismatched duplexDNAs, an MSH dimer may be associated with the detector surface of theOABS, whereby a mismatched duplex DNA binds to the homolog in thepresence of ADP and is detected, and whereby a non-mismatched duplex DNAdoes not bind appreciably to the dimer and is not detected.

[0139] Mismatched duplex DNA may be dissociated from an MSH dimer afterseparating the MSH dimer from a population comprising the mismatchedduplex DNA and non-mismatched duplex DNAs. The mechanism by which thisdissociation may be achieved depends upon whether or not the duplex DNAhas a free end.

[0140] If the duplex DNA has a free end, then the an MSH dimer may bedissociated from the duplex DNA by contacting the dimer-mismatchedduplex DNA complex with a solution having a high salt concentration,with a solution comprising EDTA or another magnesium-chelating agent, orwith a releasing solution comprising ATP. Preferably, such a releasingsolution comprises at least about 0.3 micromolar ATP, more preferably atleast about 3 micromolar, more preferably at least about 30 micromolarATP, and even more preferably much more than 30 micromolar ATP (e.g. 200micromolar ATP or 500 micromolar ATP). If the mismatched duplex DNA hasa free end, then the MSH dimer may be dissociated therefrom simply bycontacting the dimer with a solution comprising ATP. The MSH dimer mayalso be dissociated from the mismatched duplex DNA by contacting thedimer-mismatched duplex DNA complex with a gamma-modified ATP analog.

[0141] If the mismatched duplex DNA does not have a free end, then anMSH dimer may be dissociated from the duplex DNA by contacting thedimer-mismatched duplex DNA complex with a solution which comprises highsalt or EDTA or another magnesium-chelating agent. The dimer will notdissociate from the duplex DNA having no free end in the presence of ATPand magnesium ions (e.g. at least about 10 nanomolar Mg²⁺, preferably atleast about l micromolar Mg²⁺, and more preferably at least about 100micromolar Mg²⁺. However, if a free end is generated on the mismatchedduplex DNA, for example, by cleaving a circular DNA, by removing ablocking group from a blocked end of the DNA, or by cleaving the blockedend of the DNA, then the dimer will dissociate from the duplex DNA inthe presence of ATP and magnesium ions. It is understood that there maybe some situations in which association of MSH dimers is advantageous(e.g. separating DNA associated with MSH from DNA not associated withMSH). In such situations, taking advantage of the property of MSH dimersto exchange ADP to ATP only when a mismatch is present will permitassociation of multiple copies of the MSH dimer with the DNA,effectively increasing the amount of MSH dimer which can be detectedusing one or more of the methods described herein. This increase may beparticularly important where the detection limit of the assay isrelatively low.

[0142] Mismatched duplex DNA may be separated from a population ofduplex DNAs by contacting the population and an MSH dimer and bindingthe MSH dimer to a support after contacting it with the population, butprior to separating the non-mismatched duplex DNA from the MSH dimer.

[0143] It is understood that if acceleration of ATP displacement of ADPbound to an MSH dimer or acceleration of ATP hydrolysis by MSH dimer notbound to duplex DNA is desired, the MSH dimer may be contacted with aMutL homolog to achieve this acceleration. It is furthermore understoodthat if the MSH dimer is present in molar excess with respect to themismatched duplex DNA an average of more than one copy of the MSH dimermay be associated with individual copies of the mismatched duplex DNA ifATP is available to the MSH dimer. The average number of copies of theMSH dimer associated with individual copies of the mismatched duplex DNAmay be further increased by contacting the MSH dimer with a MutLhomolog. Similarly, the average number of copies of the MSH dimerassociated with individual copies of the mismatched duplex DNA may beincreased by employing solutions which favor formation of ADP-bound MSHdimer and displacement of ADP bound to mismatch-bound dimer by ATP. Suchconditions include, but are not limited to, increasing the concentrationADP in the binding solution, increasing the concentration ATP,magnesium, or both, in the binding solution, and increasing theconcentration of the dimer in the binding solution.

[0144] The properties of MSH dimers described above can be employed in avariety of useful methods including, but not limited to the following.It is understood that other methods which usefully employ the methodsdescribed above may be devised by the ordinarily skilled worker in viewof the teachings provided herein.

[0145] The invention includes a method of segregating a mismatchedduplex DNA from a population of DNA molecules. This method comprisescontacting an MSH dimer and the population in the presence of a bindingsolution comprising a nucleotide selected from the group consisting ofADP and ATP. In the presence of this binding solution, the MSH dimerassociates with the duplex DNA. After contacting the dimer and thepopulation, the MSH dimer is segregated from the population. The duplexDNA is thereby segregated from the population.

[0146] The invention also includes a method of detecting a differencebetween a sample nucleotide sequence and a reference nucleotidesequence. This method comprises annealing a first DNA strand and asecond DNA strand to form a duplex DNA. The first DNA strand has thesample nucleotide sequence, and the second DNA strand has a nucleotidesequence which is complementary to the reference nucleotide sequence. Ifthere is a difference between the sample nucleotide sequence and thereference nucleotide sequence, then the duplex DNA will be a mismatchedduplex DNA. After annealing the DNA strands, the duplex DNA and an MSHdimer are contacted in the presence of a binding solution as describedherein. If the duplex DNA is a mismatched duplex DNA, then the MSI dimerassociates with the duplex DNA. After contacting the duplex DNA and theMSH dimer, association of the MSH dimer with the duplex DNA molecule isdetected as described herein. Association of the MSH dimer with theduplex DNA molecule is an indication that there is a difference betweenthe sample nucleotide sequence and the reference nucleotide sequence.

[0147] The invention further includes a method of determining whether amammal is predisposed for carcinogenesis. This method comprisesannealing a first DNA strand and a second DNA strand to form a duplexDNA. The first DNA strand has the nucleotide sequence of at least aregion of an oncogene or a tumor suppressor gene of the mammal, such asone of those described herein. The second DNA strand has a nucleotidesequence which is complementary to the consensus nucleotide sequence ofthis region. If there is a sequence difference between the first DNAstrand and the second DNA strand, then the duplex DNA will be amismatched duplex DNA. The duplex DNA is contacted with an MSH dimer inthe presence of a binding solution as described herein. The MSH dimerassociates with the duplex DNA if the duplex DNA is a mismatched duplexDNA. After contacting the duplex DNA and the MSH dimer, association ofthe MSH dimer with the duplex DNA molecule is detected as describedherein. Association of the MSH dimer with the duplex DNA molecule is anindication that the mammal is predisposed for carcinogenesis.

[0148] The invention still further includes a method of fractionating apopulation of duplex DNAs. This method comprises contacting thepopulation with an MSH dimer in the presence of a binding solution asdescribed herein. The MSH dimer associates with any mismatched duplexDNA in the population. The MSH dimer is segregated from the population,and any mismatched duplex DNA from the population is segregated from thepopulation. The population is thereby fractionated.

[0149] The invention also includes a method of selectively amplifying atleast one mismatched duplex DNA of a population of duplex DNAs. Thismethod comprises contacting the population with an MSH dimer in thepresence of a binding solution as described herein. The MSH dimerassociates with the mismatched duplex DNA. The MSH dimer is segregatedfrom the population, and the mismatched duplex DNA is thereby segregatedfrom the population. The mismatched duplex DNA is then amplified.

[0150] The invention further includes a method of determining whetherthe nucleotide sequence of a first copy of a genomic sequence differsfrom the nucleotide sequence of a second copy of the genomic sequence.This method comprises amplifying a region of each of the first copy andthe second copy of the genomic sequence to yield amplified first copiesand amplified second copies. The amplified first copies and theamplified second copies are mixed and denatured to form a first mixture.The nucleic acids in the first mixture are annealed to form a secondmixture comprising duplex DNAs. If the nucleotide sequence of first copyand the nucleotide sequence of the second copy of the genomic sequencediffer, then at least some of the duplex DNAs in the second mixture aremismatched duplex DNAs. The second mixture is contacted with an MSHdimer in the presence of a binding solution as described herein. The MSHdimer associates with any mismatched duplex DNAs that are present in thesecond mixture. Association of the MSH dimer with duplex DNA is thendetected. Association of the MSH dimer duplex DNA is an indication thatthe nucleotide sequence of the first copy of the genomic sequencediffers from the nucleotide sequence of the second copy of the genomicsequence. The first and second copies of the genomic sequence may beobtained from a single eukaryotic organism or from different eukaryoticindividuals of the same or a different species. If the first and secondcopies of the genomic sequence are obtained from a single individual,one copy may be obtained from each of a pair of the individual'schromosomes. If the first and second copies of the genomic sequence areobtained from different individuals of the same species, then theindividuals may, for example, be related, unrelated, or congenic.

[0151] The invention yet further includes a composition for segregatinga mismatched duplex DNA from a population of duplex DNAs, thecomposition comprises an MSH dimer bound to a support, and may be usedin any of the methods described herein. The composition may be acomponent of a kit which includes an instructional material whichdescribes a method of the invention wherein the composition is useful.The kit may instead comprise the composition and a binding solution or areleasing solution, as described herein.

[0152] The invention also includes a kit for screening a genomic regionfor a nucleotide sequence which differs from a reference nucleotidesequence. This kit comprises a pair of primers complementary to the endsof the region. The pair of primers is useful for amplifying the region.The kit further includes a DNA strand having the reference nucleotidesequence and at least one MutS homolog. The MutS homolog may be suppliedin the form of an MSH dimer. The kit may be used to perform the methodsdescribed herein. The kit may further comprise additional components,such as an instructional material which describes use of the kit toperform a method described herein, an assay reagent for detectingbinding of a mismatched duplex DNA to the MSH dimer, or a reagent forblocking the ends of duplex DNAs. By way of example, the primers of thekit may be biotinylated and the kit may further comprise an avidin suchas streptavidin for blocking the ends of duplex DNA.

[0153] The invention further includes a kit for separating a mismatchedduplex DNA from non-mismatched duplex DNAs. This kit comprising at leastone MutS homolog, a linker for binding the MutS homolog to a support,and an additional reagent selected from the group consisting of anucleotide and a releasing solution, as described herein. The releasingsolution may, for example, comprise a compound selected from the groupconsisting of ATP and a gamma-modified ATP analog. The kit may furthercomprise a reagent for blocking the ends of a duplex DNA, such asbiotinylated PCR primers which can be used to amplify the duplex DNA,prior to contacting the biotinylated duplex DNA with an avidin such asstreptavidin. Alternately, the kit may comprise a binding solution whichis substantially free of ATP, magnesium ions, or both, whereby when asupport-bound MSH dimer binds a mismatched duplex DNA, the dimer is notable to bind ATP and magnesium ion, and thus cannot exhibitDNA-associated diffusion and the duplex DNA remains bound to theADP-bound dimer.

[0154] The invention further includes a nonhuman mammal which isnullizygous for both Msh2 and p53. The nonhuman mammal does not expressMsh2 or p53 and exhibits a phenotype selected from the group consistingof inappropriate fetal apoptosis and a predisposition forcarcinogenesis. Preferably, the mammal is a mouse, but other non-humanmammals may also be generated using the teaching provided herein.

[0155] The invention still further includes a method of making anonhuman mammal which is nullizygous for both Msh2 and p53. Such amammal does not express Msh2 or p53 and exhibits a phenotype selectedfrom the group consisting of a predisposition for inappropriate fetalapoptosis and a predisposition for carcinogenesis. Such mammals are madeby mating a first parent mammal comprising at least one null allele ofMsh2 and at least one null allele of p53 and a second parent mammalcomprising at least one null allele of Msh2 and at least one null alleleof p53. The offspring of the two parent mammals inherit the null allelesof these two genes according to normal allelic segregation rules (i.e.generally speaking, most mammals will randomly inherit one of eachparent's two alleles of a gene). Thus, the proportion of nonhumanmammals which are nullizygous for both Msh2 and p53 will depend upon theallelic composition of the parents. Offspring which are nullizygous forboth Msh2 and p53 do not express Msh2 or p53 and exhibit a phenotypeselected from the group consisting of inappropriate fetal apoptosis anda predisposition for carcinogenesis. Further details relating to thismethod are described herein, such as in Example 2.

[0156] The invention also includes several screening methods, all ofwhich make use of the properties of the Msh2^(−/−) p53^(−/−) micedescribed herein.

[0157] A standard screening procedure is now described which is usefulfor determining the tumorigenesis-, apoptosis-, aging-, or fetaldevelopment-modulating potential of a compound. While this procedures isdescribed with respect to particular protocols and mice, it will beappreciated that the screening procedure described should not beconstrued to limit the invention in any way.

[0158] Msh2^(−/−) p53^(−/−) mice are generated as described herein orobtained from a producer of such mice. A predetermined amount of thecompound is administered to a Msh2^(−/−)p53^(−/−)mouse by any practicalmeans. The method of administration of the compound is not critical. Byway of example, the compound may be administered orally,intraperitoneally, intravenously, topically, intramuscularly, or via apulmonary route.

[0159] Following administration of the compound, the Msh2^(−/−)p53^(−/−)mouse, each Msh₂ ^(−/−) p53^(−/−) mouse is observed for aboutfour months. Each mouse is examined approximately daily. Every week,each mouse is weighed, observed for any clinically-relevant symptoms,and the number and extent of tumors are assessed.

[0160] To reduce any potential for bias, the study is blinded. A firstinvestigator treats all mice with compound(s) and identifiably marks orcages the transgenic mice, so that the nature of the treatments will notbe known to a second investigator, who performs all tumor counts,weighing, and general observations.

[0161] If the mice are being used to screen for tumorigenesis-modulatingcompounds, then after observations are completed, the rate of tumorincidence and the tumor yield are determined for each group of M mice towhich the compound was applied. A higher or lower rate of tumorincidence or a higher or lower tumor yield for a group of Msh2−/−p₅₃^(−/−) mice to which the compound was applied, compared with the levelsof tumor incidence and tumor yield for a group of Msh2^(−/−)p53 ^(−/−)mice to which the compound was not applied, is an indication that thecompound affects tumorigenesis.

[0162] If the mice are being used to screen for apoptosis-modulatingcompounds or fetal development-modulating compounds, then the mice arepreferably administered the compound and observed during fetaldevelopment. After observations are completed, the prevalence ofinappropriate fetal apoptosis and the fetal survival rate are determinedfor each group of Msh2^(−/−)p53^(−/−) mouse embryos to which thecompound was applied. A higher or lower mouse embryos or a higher orlower fetal survival rate for a group of Msh2^(−/−)p53^(−/−) mouseembryos to which the compound was applied, compared with the mouseembryos and fetal survival rate for a group of Msh2^(−/−)p53^(−/−) mouseembryos to which the compound was not applied, is an indication that thecompound affects apoptosis or fetal development.

[0163] If the mice are being used to screen for aging-modulatingcompounds, then after observations are completed, the prevalence of atleast one symptom of aging (e.g. graying of hair, other changes in coatcolor, lethargy, or hair loss) are determined for each group ofMsh2^(−/−)p53^(−/−) mice to which the compound was applied. A higher orlower prevalence of a symptom of aging for a group ofMsh2^(−/−)p53^(−/−) mice to which the compound was applied, comparedwith the prevalence of the symptom for a group of Msh2^(−/−)p53^(−/−)mice to which the compound was not applied, is an indication that thecompound affects aging.

[0164] Preferably, groups of Msh2^(−/−)p53^(−/−) mice or embryos areused, with each mouse in a group being treated identically. Alsopreferred are studies in which one of at least three different doselevels of the compound are applied to the mice or embryos in each of atleast three corresponding groups of transgenic mice. It is preferred,where possible, to demonstrate a statistically significant difference(P<0.05) between the observed phenotype for the first dose level and theobserved phenotype for the third dose level.

[0165] A cell line may be made using cells obtained from a Msh2^(−/−)p53^(−/−) mouse of the invention. Methods of making a cell line from acell of a nonhuman animal are well known in the art.

[0166] The invention also includes a method of determining whether acomposition interferes with the activity of one of the p53 gene or oneof its expression products and a MutS homolog gene or one of itsexpression products. According to this method, non-human mammals such asmice are generated which are nullizygous for one of the p53 gene and thegene encoding the MutS homolog. These nullizygous animals are crossed togenerate embryos which are also nullizygous for the same gene. Theembryos are contacted with the composition, either in vitro or in utero,and the effects of contacting the embryos with the composition areobserved. Increased mortality among the embryos, particularly among thefemale embryos, is an indication that the composition is able tointerfere with the activity of the other of the p53 gene or one of itsexpression products and a MutS homolog gene or one of its expressionproducts. Thus, the ability of a composition to increase femaleembryonic lethality in mouse embryos which are nullizygous for the p53gene is an indication that the composition interferes with the activityof a MutS homolog gene or one of its expression products. Similarly, theability of a composition to increase female embryonic lethality in mouseembryos which are nullizygous for a MutS homolog gene is an indicationthat the composition interferes with the activity of the p53 gene or oneof its expression products. Preferably, female embryos are selected andused. Also preferably, female embryonic lethality is observed at about9.5 days gestation. Methods of generating both nullizygous p53 animalssuch as mice and nullizygous msh gene animals such as mice have beendescribed in the art.

[0167] The invention further includes a composition comprising a humanMutS homolog fragment, wherein the fragment comprises a MutS homologinteraction region. The fragment may be a polypeptide having as many asall but one amino acid residues of the corresponding MutS homolog. Theinteraction region may be any of the MutS homolog interaction regionsdescribed herein or a MutS homolog interaction region having significanthomology thereto. By way of example, a MutS homolog interaction regionhaving significant homology to a MutS homolog interaction regiondescribed herein may exhibit at least about 50%, and preferably at leastabout 70%, 85%, 95%, or 99% homology with a MutS homolog interactionregion described herein. Thus, by way of example, the interaction regionmay be completely or significantly homologous to amino acid residues378-625 of hMSH2, amino acid residues 875-934 of hMSH2, amino acidresidues 126-250 of hMSH3, amino acid residues 1050-1128 of hMSH3, aminoacid residues 326-575 of hMSH6, or amino acid residues 1302-1360 ofhMSH6.

[0168] The composition comprising a human MutS homolog fragment of theinvention is useful in a method of inhibiting association of a firsthuman MutS homolog and a second human MutS homolog. This methodcomprises contacting at least one of the first human MutS homolog andthe second human MutS homolog with the human MutS homolog fragment ofthe invention. Without wishing to be bound by any particular theory ofoperation, it is believed that the fragment will interact with at leastone interaction region of one human MutS homolog, thereby preventingthat homolog from associating with the other MutS homolog. Suchcompounds would have utility for inducing apoptosis in animal cells(e.g. human tumor cells) which harbor one or more mutations in their p53genes. Such compounds would also be useful for sensitizing animal cellswhich harbor one or more mutations in their p53 genes for furthertreatment using, for example, DNA-damaging agents.

[0169] As described herein in Example 5, cDNA encoding hMSH5 has beendiscovered, and a protein encoded by that cDNA has also been discovered.hMSH5 may be purified in a manner directly analogous to the methodsdescribed herein (e.g. by his-tagging) or by other methods well known inthe art. The invention thus includes substantially purified hMSH5 and anisolated nucleic acid encoding hMSH5.

[0170] The invention is now described with reference to the followingExamples. These Examples are provided for the purpose of illustrationonly and the invention should in no way be construed as being limited tothese examples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

EXAMPLE 1

[0171] The Human Mismatch Recognition Complex hMSH2:hMSH6 Functions as aMolecular Switch

[0172] Adenine nucleotide binding by the human hMSH2:hMSH6 mismatchrecognition complex functions as a novel molecular switch. ThehMSH2:hMSH6 heterodimer is “ON” (i.e. it associates with mismatched DNA)in the ADP-bound form, and “OFF” (i.e. it is not capable of associatingwith mismatched DNA with which it is not already associated) in theATP-bound form. The data presented herein establish that the switch is‘turned OFF’ by displacement of complex-bound ADP by ATP. ATP-boundcomplex is recycled to the ADP-bound form, which is capable of bindingto mismatched DNA, by intrinsic ATPase activity of the complex.

[0173] The materials and methods used in the experiments presented inthis Example are now described.

[0174] Overexpression and purification of hMSH2:hMSH6

[0175] Clones encoding hMSH2 and those encoding hMSH6 have beendescribed (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA93:13629-13634; Fishel et al., 1993, Cell 75:1027-1038). In theexperiments described herein, the clone encoding hMSH6 was modified tofurther encode six histidine residues at the amino terminus of the hMSH6protein molecule. hMSH3 can be similarly modified and isolated.

[0176] hMSH2 and hMSH6 were overexpressed in SF9 insect cells using thepFastBac™ dual expression vector (Gibco BRL, Grand Island, N.Y.). asdescribed n the BAC-TO-BAC™ baculovirus expression systems protocol(Gibco BRL, Grand Island, N.Y.). Briefly, SF9 cells suspended inapproximately 400 milliliters culture medium were infected using thevector, and were then cultured for 48 hours to achieve a cell density ofapproximately 10⁶ SF9 cells per milliliter. The cells contained in 200milliliter aliquots of SF9 cells were harvested by centrifugation at 200x g, resuspended in 10 milliliters of buffer A, and frozen at −80° C.Buffer A comprised 300 millimolar NaCl, 20 millimolar imidazole, 25millimolar HEPES buffer adjusted to pH 7.8 using NaOH, 10% (v/v)glycerol, 0.5 millimolar phenylmethylsulfonylfluoride (PMSF), 0.8micrograms per milliliter pepstatin, and 0.8 micrograms per milliliterleupeptin.

[0177] Cell extracts were prepared by thawing the cells, passing thecells through a 25 gauge needle, and then ultracentrifuging the extractat 40,000 rotations per minute in a Beckman Ti60 rotor for 70 minutes,according to known methods. About 100 milliliters of infected cellsyielded approximately 2 milligrams of hMSH2:hMSH6 protein complex. Allof the following protein purification procedures in this Example werecarried out at 4° C.

[0178] The supernatant was applied to a 2 milliliter nickel-NTASUPERFLOW™ column (Qiagen, Chatsworth, Calif.) at a flow rate of 0.15milliliters per minute using a Pharmacia FPLC system. The column waswashed by passing 35 milliliters of buffer A through the column. Afterwashing the column, the hMSH2:hMSH6 heterodimer was eluted by applying30 milliliters of buffer A comprising a linear gradient of imidazole tothe column and collecting the eluent from the column in fractions,wherein the concentration of imidizole was varied from 20 millimolar to200 millimolar. The hMSH2:hMSH6 heterodimer eluted in fractionscontaining approximately 70 millimolar imidizole.

[0179] Fractions from the nickel-NTA column which contained peak amountsof the heterodimer were loaded at a flow rate of 0.2 milliliters perminute directly onto a 1 milliliter PBE 94 column (a polybuffer exchangecolumn obtained from Pharmacia, Upsala Sweden) which had beenequilibrated with buffer B. Buffer B comprised 300 millimolar NaCl, 25millimolar HEPES buffer adjusted to pH 7.8 using NaOH, 1 millimolardithiothreitol (DTT), 0.1 millimolar ethylenediaminetetraacetic acid(EDTA), 10% (v/v) glycerol, 0.5 millimolar PMSF, 0.8 micrograms permilliliter pepstatin, and 0.8 micrograms per milliliter leupeptin. ThePBE 94 column was washed by passing 10 milliliters of buffer B throughthe column. After washing the column, the hMSH2/hMSH6 complex was elutedby applying 20 milliliters of buffer B comprising a linear gradient ofNaCl to the column and collecting the eluent from the column infractions, wherein the concentration of NaCl was varied from 300millimolar to 1 molar. The hMSH2:hMSH6 heterodimer eluted from the PBE94 column in fractions containing approximately 575 millimolar NaCl.

[0180] Fractions collected from the PBE 94 column which contained peakamounts of the heterodimer were dialyzed twice for two hours against 2liters of a solution comprising 100 millimolar NaCl, 25 millimolar HEPESbuffer adjusted to pH 7.8 using NaOH, 1 millimolar DTT, 0.1 millimolarEDTA, and 20% (v/v) glycerol. Aliquots of the dialyzed solutioncontaining the heterodimer were frozen using liquid nitrogen and storedat −80° C. for several months without detectable loss of activity.

[0181] hMSH2, hMSH6, and bovine serum albumin (BSA) contain nearlyidentical percentages (12%, 14%, and 13%, respectively) of arginine andheterocyclic amino acids, the amino acids known to interact with theCoomassie Brilliant Blue stain. Protein concentration in an aliquotcomprising the hMSH2:hMSH6 heterodimer was determined by subjecting aportion of the aliquot to SDS-PAGE using a 6% (w/v) acrylamide gel,subjecting a known amount of BSA (Boehringer Mannheim, Indianapolis,Ind.) to SDS-PAGE using a 6% (w/v) acrylamide gel, staining the SDS-PAGEgels with Coomassie Brilliant Blue, and comparing the intensities of theprotein bands in the gels to a BSA standard on a Coomassie stained 6%SDS PAGE to calculate protein concentration. The intensities of stainedprotein bands were measured using BioRad Gel Doc and MOLECULAR ANALYST™software. This protein quantitation method revealed the hMSH2 and hMSH6proteins to be in near exact equimolar proportion in the heterodimer.Preparation of 39- and 81-base pair oligonucleotide probes

[0182] The sequence of the 39-base pair oligonucleotide used in theexperiments presented in this Example was: 5′-CGGCGAATTC CACCAAGCTTGATCGCTCGA GGTACCAGG-3′ (SEQ ID NO: 1). The homologous 39-base pair DNAsubstrate used in the experiments presented in this Example was made byannealing the 39-base pair oligonucleotide with an oligonucleotide (SEQID NO: 2) which was completely complementary thereto. The G/T mismatched39-base pair DNA substrate used in the experiments presented in thisExample was made by annealing the 39-base pair oligonucleotide with anoligonucleotide (SEQ ID NO: 3) which was completely complementarythereto, except that the oligonucleotide contained a G residue at thenucleotide position complementary to the T residue at position 20(numbered in the direction extending from the 5′ end to the 3′ end) ofthe 39-base pair oligonucleotide. SEQ ID NO: 2 and SEQ ID NO: 3 arelisted in FIG. 8.

[0183] The nucleotide sequence of the 81-base pair oligonucleotide usedin the experiments described in this Example was: 5′-AAAGCTGGAGCTGAAGCTTA GCTTAGGATC ATCGAGGATC GAGCTCGGTG CAATTCAGCG GTACCCAATTCGCCCTATAG T-3′ (SEQ ID NO: 4). The homologous 81-base pair DNAsubstrate used in the experiments presented in this Example was made byannealing the 81-base pair oligonucleotide with an oligonucleotide (SEQID NO: 5) which was completely complementary thereto. The G/T mismatched81-base pair DNA substrate used in the experiments presented in thisExample was made by annealing the 81-base pair oligonucleotide with anoligonucleotide (having the nucleotide sequence listed in SEQ ID NO: 6)which was completely complementary thereto, except that theoligonucleotide contained a T residue at the nucleotide positioncomplementary to the G residue at position 41 (numbered in the directionextending from the 5′end to the 3′ end) of the 81-base pairoligonucleotide. SEQ ID NO: 5 and SEQ ID NO: 6 are listed in FIG. 8.

[0184]³²P-end-labeled DNA substrates were prepared by incubating singlestranded oligonucleotides in the presence of T4 polynucleotide kinase(Promega Corp., Madison, Wis.) and [³²P]gamma-ATP (NEN Dupont,Wilmington, Del.). Excess label was separated from the labeled DNAsubstrates using a CENTRISEP™ column (Princeton Separations, Princeton,N.J.) per the manufacturer's instructions.

[0185] Labeled DNA substrate was annealed with a single-stranded DNAmolecule which was either completely complementary thereto or containeda single G/T mismatch. To anneal the labeled DNA substrate with thesingle-stranded DNA molecule, the labeled molecule was suspended in asolution comprising a 10-fold excess of the single-stranded DNAmolecule, 10 millimolar Tris buffer which had been adjusted to pH 7.5using HCl, 100 millimolar NaCl, and 1 millimolar EDTA. The suspensionwas heated to 95° C. and then slowly cooled to 55° C. and was maintainedat this temperature for twelve hours. Single-stranded DNA was removedfrom the suspension by incubating the suspension with benzoylatednaphthoylated DEAE cellulose (BND cellulose, Sigma Chemical Co., St.Louis, Mo.) for twenty minutes in the presence of a solution comprising1.5 molar NaCl, 20 millimolar Tris buffer which had been adjusted to pH7.5 using HCl, and 0.5 millimolar EDTA. BND cellulose was then pelletedby centrifuging the suspension for about five minutes using an Eppendorfbench-top centrifuge. Double-stranded DNA, which remained in thesupernatant, was separated from the BND cellulose by filtration and wasthen precipitated by adding ethanol to the supernatant. Thedouble-stranded labeled DNA substrate was resuspended in a solutioncomprising 10 millimolar Tris buffer which had been adjusted to pH 7.5using HCl, 100 millimolar NaCl, and 1 millimolar EDTA. Single-strandedDNA could not be detected in the solution, as assessed by 4% (w/v)native PAGE separation of the nucleotides in the solution.Non-³²P-labeled oligonucleotides were prepared using analogous methods.

[0186] Gel Mobility Shift Assays

[0187] Gel mobility shift assays were performed by incubating ahMSH2:hMSH6 heterodimer and 9 femtomoles of either the ³²P-labeledhomologous 81-base pair DNA substrate or the ³²P-labeled G/T-mismatched81-base pair DNA substrate in a buffer comprising 50 millimolar NaCl, 25millimolar HEPES buffer which had been adjusted to pH 7.5 using NaOH, 1millimolar DTT, 0.01 millimolar EDTA, and 15% (v/v) glycerol. The bufferincluded 10 nanograms per microliter of poly dI-dC (Pharmacia LKBBiotechnology Inc., Piscataway, N.J.). Poly dI-dC is an alternatingnucleic acid polymer which does not interfere with binding of thehMSH2:hMSH6 heterodimer to DNA. In certain experiments described herein,the incubation mixture further comprised selected concentrations ofnucleotides or non-labeled DNA. In other experiments described herein,the incubation mixture further comprised 1 millimolar MgCl₂ or 5millimolar EDTA. Except as otherwise described herein, each incubationmixture had a volume of 20 microliters and was incubated for fifteenminutes at 37° C. and then immediately placed on ice. Each incubationmixture was applied to a gel comprising 4% (w/v) polyacrylamide (29:1ratio of acrylamide:bis-acrylamide) 4% (v/v) glycerol, 40 millimolarTris acetate buffer (pH 7.8), and 1 millimolar EDTA. Electrophoresis wasperformed by applying 200 volts to the gel for two hours. Followingelectrophoresis, each gel was dried and quantitated using aphosphoimaging device obtained from Molecular Dynamics.

[0188] Footprint Assays

[0189] Incubation of the hMSH2:hMSH6 heterodimer with ³²P-labeled DNAsubstrates was performed as described for gel mobility shift assays,except that 18 femtomoles of 32p-labeled DNA substrate was used in eachassay. Following incubation, 80 microliters of a buffer comprising 50millimolar NaCl, 25 millimolar HEPES buffer which had been adjusted topH 7.8 using NaOH, 1 millimolar DTT, 10 nanograms per microliter polydI-dC, 1.25 millimolar CaCl₂, 3.1 millimolar MgCl₂, 10% (v/v) glycerol,and 33 picograms per microliter DNase (Boehringer Mannheim,Indianapolis, Ind.) was added to each incubation mixture. The mixtureswere incubated at 37° C. for an additional three minutes, and then 0.7milliliters of a solution having a pH of 5.2 and comprising 95% (v/v)ethanol and 180 millimolar sodium acetate was added to each mixture tohalt the DNase reaction and to precipitate the nucleic acids present inthe mixture.

[0190] DNase-treated nucleic acids were resuspended in 4 microliters ofa solution comprising 80% (v/v) formamide, 10 millimolar NaOH, 1millimolar EDTA, and 0.1% (w/v) bromophenol blue. The suspension washeated at 90° C. for five minutes and was applied to a gel comprising 8%(w/v) polyacrylamide (29:1 ratio of acrylamide:bis-acrylamide), 90millimolar tris-borate buffer (pH 8), and 2 millimolar EDTA. Followingelectrophoresis for 2 hours at 200 volts, each gel was dried and imagedon a phosphoimaging device. Individual bases of the 81-base pair DNAsubstrates were identified by Maxam-Gilbert sequencing reactionsperformed as described (Ausubel et al., 1994, Current Protocols inMolecular Biology, 8th Ed., Janssen, ed., John Wiley & Sons, Inc.,Boston).

[0191] ATPase Assays

[0192] ATPase activity was measured in a reaction mixture comprising 20microliters of Buffer P, 500 micromolar non-labeled ATP (except whereindicated), and 16.5 nanomolar [⁻³²P]gamma-ATP. Buffer P comprised 40millimolar HEPES which had been adjusted to pH 7.8 using NaOH, 75millimolar NaCl, 10 millimolar MgCl₂, 1.75 millimolar DTT, and 0.075millimolar EDTA, and 15% (v/v) glycerol. Steady state reactionmeasurements were made using 60 nanomolar hMSH2:hMSH6 heterodimer andeither 240 nanomolar homoduplex 39-base pair DNA substrate or 240nanomolar G/T mismatched 39-base pair DNA substrate. Reaction mixtureswere incubated at 37° C. for thirty minutes, and the reaction wasstopped by addition of 400 microliters of a solution comprising 10%(w/v) activated charcoal (Sigma Chemical Co., St. Louis, Mo.) and 1millimolar EDTA. Charcoal was pelleted by centrifuging the mixture at10,000 rotations per minute for ten minutes. The 32p content ofduplicate 100 microliter aliquots of the supernatant was assessed byliquid scintillation.

[0193] Initial velocity measurements were made by incubating thehMSH2:hMSH6 heterodimer for ten minutes at 25° C. in a reaction mixturecomprising one volume Buffer P containing no MgCl₂, 200 nanomolarnon-labeled ATP, and 16.5 nanomolar [³²P]gamma-ATP. To start thereaction, an equal volume of buffer P comprising 20 millimolar MgCl₂ and1 millimolar non-labeled ATP was mixed with the reaction mixture, whichraised the MgCI₂ and ATP concentrations to 10 millimolar and 500micromolar, respectively. Aliquots were removed at selected times andelectrophoresed as described herein. A control aliquot was removed andprepared for electrophoresis prior to addition of the MgCl₂-containingBuffer P to the reaction mixture.

[0194] ADP Exchange Assays

[0195] The ADP-ATP exchange rate was determined in a reaction mixturewhich comprised Buffer Q, 2.3 micromolar [³H]-ADP, and 60 nanomolarhMSH2:hMSH6 heterodimer. Buffer Q comprised 25 millimolar HEPES whichhad been adjusted to pH 7.8 using NaOH, 75 millimolar NaCl, 10millimolar MgCl₂, 1 millimolar DTT, and 15% (v/v) glycerol. Thisreaction mixture was incubated for ten minutes at room temperature. 240nanomolar G/T-mismatched 39-base pair DNA substrate was added to thereaction mixture, and the incubation was continued for an additional tenminutes. The final volume of the reaction mixture was 10 microliters.The order of addition of DNA and ADP did not affect the kinetic resultsobtained using this assay. An equal volume Buffer Q comprising 1millimolar non-labeled ATP was then added to the reaction mixture.Reactions were incubated at 25° C. for a selected time and then haltedby diluting the reaction mixture with 4 milliliters of an ice-cold stopbuffer comprising 25 millimolar HEPES which had been adjusted to pH 7.8using NaOH, 100 millimolar NaCl, and 10 millimolar MgCl₂.

[0196] Each halted reaction mixture was immediately filtered on a HAWPnitrocellulose membrane (Millipore, Bedford, Mass.) and washed thricewith 4 milliliters of the ice-cold stop buffer. Each filter was airdried and incubated overnight in a standard scintillation cocktail.Radioactivity retained on the filters was quantified using a Beckmanscintillation counter. A control reaction mixture was prepared by notadding the Buffer Q comprising 1 millimolar non-labeled ATP to thereaction mixture. The amount of [³H]-ADP retained on the membrane towhich the control reaction mixture was applied was considered tocorrespond to the amount of radioactivity retained when 100% of thecomplex had [³H]-ADP bound thereto.

[0197] Thin Layer Chromatography (TLC) Analysis

[0198] TLC was used to determine the composition of an ATPase reactionmixture which was prepared as described herein in the presence of theG/T-mismatched 39-base pair DNA substrate, 15 micromolar ATP, and 0.0lmicromolar [³²P]alpha-ATP and which was permitted to react for twentyminutes at 37° C. TLC was performed as previously described (Fishel etal., 1988, Proc. Natl. Acad. Sci. USA 85:36-40).

[0199] The results of the experiments presented in this Example are nowdescribed. Overexpression and purification of the hMSH2-hMSH6 proteincomplex

[0200] hMSH2 and hMSH6 proteins were overexpressed in insect cells usinga dual expression baculovirus vector, as assessed by the SDS-PAGEanalysis of proteins obtained from cell extract. Co-expression of hMSH2and hMSH6 proteins resulted in formation of a completely solublehMSH2:hMSH6 heterodimer. Independent expression of either protein aloneresulted in formation of a substantial amount of insoluble proteinproduct. hMSH2 and hMSH6 likely exist together as a highly stablecomplex in vivo, as judged by the results obtained in the experimentsdescribed in this Example, the ability of investigators to co-purifythese two proteins from human cells (Drummond et al., 1995, Science268:1909-1912), and the ability of these two proteins to interact invitro (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634).

[0201] Purification of hMSH2 and hMSH6 from insect cells indicated thata stable heterodimer of the two proteins had been formed. Quantitativedensitometry of Coomassie-stained products consistently revealed thatthe hMSH2 and hMSH6 subunits were present in an equimolar ratio, as wasobserved with the yeast MSH2:MSH6 protein complex (Alani et al., 1997,Mol. Cell Biol. 17: 2436-2447). The purification methodology describedherein yielded a protein preparation which was more than 95%homogeneous, which exhibited high MSH2MSH6 activity, and which appearedto be free of any contaminating nucleic acid or nucleotide.

[0202] G/T mismatch binding by hMSH2:hMSH6 is a model for mismatchrecognition

[0203] The hMSH2:hMSH6 heterodimer has been demonstrated herein and byothers to bind to the eight possible mismatched nucleotide combinations,as well as to a subset of single nucleotide insertion/deletionmismatches (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA93:13629-13634; Drummond et al., 1995, Science 268:1909-1912; Hughes etal., 1992, J. Biol. Chem. 267:23876-23882). The G/T mismatch was chosenas a model for quantitative analysis of hMSH2:hMSH6 mismatch bindingbecause of its apparently intermediate-to-high recognition specificity,as indicated, for example, by the data presented in FIGS. 1A-1D.

[0204] The apparent dissociation constant (K_(d)) was determined in asimple buffer system comprising neither an adenine nucleotide normagnesium using the homologous 81-base pair DNA substrate and theG/T-mismatched 81-base pair DNA substrate described herein. Resultsobtained using both gel shift assays, as depicted in FIG. 1A, and DNasefootprint assays, as depicted in FIG. 1C, indicated that K_(d) of thehMSH2:hMSH6 heterodimer for G/T mismatches was 20±5 nanomolar. Bindingof non-mismatched DNA to the heterodimer was not saturable, even athomoduplex concentrations greater than 400 nanomolar.

[0205] The binding of the hMSH2:hMSH6 heterodimer to a G/T mismatch isat least ten times more efficient than binding of hMSH2 alone to the G/Tmismatch (Fishel et al., 1994, Science 266:1403-1405; Fishel et al.,1994, Cancer Res. 54:5539-5542; Mello et al., 1996, Chemistry & Biology3:579-589). This observation indicates that formation of the hMSH2:hMSH6heterodimer enhances both the affinity and the specificity ofhMSH2-binding to mismatched DNA (Acharya et al., 1996, Proc. Natl. Acad.Sci. USA 93:13629-13634).

[0206] Gel mobility shift assays performed using the G/T-mismatched39-base pair DNA substrate described herein or using the G/T-mismatched81-base pair DNA substrate and a buffer comprising 2 millimolar MgCl₂yielded results similar to those shown in FIG. 1A. The hMSH2:hMSH6heterodimer appears to bind G/T mismatched DNA in multiple forms whichare differentiable by gel mobility shift assay.

[0207] DNase footprint analysis of hMSH2:hMSH6 heterodimer binding tothe G/T-mismatched 81-base pair DNA substrate indicated that the complexasymmetrically protects about 25 nucleotides on both strands of thesubstrate. As shown in FIG. 1C, there appeared to be two domainsprotected by the complex from cleavage by DNase. One domain appeared tobe centered on the G/T mismatch in the substrate. The other domain wasadjacent the domain centered on the G/T mismatch and was separated fromthat domain by a single DNase-sensitive nucleotide. These data arequalitatively similar to those observed in similar experiments using theE. coli and T. aquaticus MutS proteins (Su et al., 1986, Proc. Natl.Acad. Sci., USA 83:5057-5061; Su et al., 1988, J. Biol. Chem.263:6829-6835; Biswas et al., 1997, J. Biol. Chem. 272:13355-13364).

[0208] Although a shifted complex could be detected by gel mobilityshift assay using homoduplex DNA, no specific DNase footprint could beidentified, as indicated by the data presented in FIG. 1D. Lack ofsaturatability and lack of a specific footprint are consistent with theability of the hMSH2:hMSH6 heterodimer to weakly and non-specificallyassociate with homoduplex DNA.

[0209] Shifted complexes formed between the heterodimer and homoduplexDNA and those formed between the heterodimer and G/T-mismatched DNAmigrated differently in gel mobility shift assays, as shown in FIGS. 1Aand 1B. Homoduplex DNA-bound heterodimer (designated ‘NS’ for‘non-specific’ in FIG. 1B) migrated more slowly than G/T-mismatchedDNA-bound heterodimer (designated ‘S’ for ‘specific’ in FIG. 1A). Theseresults suggest that homoduplex DNA-bound heterodimer adopts a differentconformation than mismatched DNA-bound heterodimer. Alternatively, theremay have been a greater quantity of the heterodimer bound to homoduplexDNA than to mismatched DNA.

[0210] When the homoduplex 39-base pair DNA substrate described hereinwas contacted with the heterodimer, no NS product was observed in thegel mobility shift assay. The DNA length dependence of NS productformation may result if a minimum number of base pairs were necessary toassume an alternative DNA and/or hMSH2- or hMSH6-protein conformation orto bind multiple hMSH2:hMSH6 heterodimers.

[0211] These results demonstrate the high specificity of heterodimerbinding to the G/T-mismatched 81-base pair DNA substrate. The bindingwas found to be quantitatively similar by both gel mobility shift andfootprint analysis. In addition, a low level non-specific binding toduplex DNA was observed and found to be easily distinguished via itsaltered mobility using gel mobility shift analysis.

[0212] The hMSH2:hMSH6 Heterodimer Converts ATP to ADP in the Presenceof Mismatched DNA

[0213] Both bacterial and yeast MutS homologs have been shown to possessintrinsic low-level ATPase activity (Alani et al., 1997, Mol. Cell Biol.17: 2436-2447; Chi et al., 1994, J. Biol. Chem. 269: 29993-29997; Chi etal., 1994, J. Biol. Chem. 269:29984-29992; Habe et al., 1988, J.Bacteriol. 170:197-202). There are conflicting reports regarding thecapacity of mismatched heteroduplex and/or homoduplex DNA to stimulatethis intrinsic ATPase activity (Alani et al., 1997, Mol. Cell Biol. 17:2436-2447; Chi et al., 1994, J. Biol. Chem. 269: 29993-29997; Chi etal., 1994, J. Biol. Chem. 269:29984-29992).

[0214] It was demonstrated in the experiments described in this Examplethat the hMSH2:hMSH6 heterodimer possesses intrinsic DNA-dependentATPase activity that is dependent upon the presence of magnesium as acofactor. Saturation of the ATPase activity by hMSH2:hMSH6 heterodimerwhich was observed at protein concentrations above 0.6 micromolar waslikely the result of a limiting amount of DNA, which was use at a fixedconcentration of 240 nanomolar in the assay. Thin layer chromatographyrevealed that hMSH2:hMSH6 heterodimer ATPase activity uniformly convertsATP to ADP and inorganic phosphate. Using Lineweaver-Burk analysis andEadie-Hofstee analysis, it was determined that hMSH2:hMSH6 heterodimerATPase is most active in the presence of a G/T mismatch. The value ofk_(cat) using ATP and G/T-mismatched DNA as substrates was about 26minute-¹. The value of K_(m) using ATP and G/T-mismatched DNA assubstrates was about 46 micromolar. hMSH2:hMSH6 heterodimer ATPase issubstantially less active in the presence of homoduplex DNA. The valueof k_(cat) using ATP and G/C-mismatched DNA as substrates was about 7.4minute-¹. The value of Km using ATP and G/C-mismatched DNA as substrateswas about 23 micromolar. hMSH2:hMSH6 heterodimer ATPase is substantiallyinactive in the absence of DNA. The value of k_(cat) using ATP alone asa substrate was about 0.9 minute¹. The value of K_(m) using ATP alone asa substrate was about 10 micromolar.

[0215] ATPase activity stimulation was the same regardless of whetherthe homoduplex DNA had a length of 39 base pairs, 81 base pairs or 2,900base pairs, and was also the same regardless of whether the mismatchedDNA had a length of 39 base pairs or 81 base pairs. These resultsindicated that hMSH2:hMSH6 heterodimer ATPase activity is not dependentupon DNA length.

[0216] It was observed that k_(cat) using ATP alone as a substrate waslower than k_(cat) using ATP and homoduplex DNA as a substrate and thisvalue was lower than k_(cat) using ATP and mismatched DNA as substrates.However, K_(m) for ATP in the absence of DNA was lower than K_(m) forATP in the presence of homoduplex DNA, and this value was lower thanK_(m) for ATP in the presence of mismatched DNA. These observationsindicated that although the rate of hydrolysis is increased in thepresence of a mismatch, the affinity for ATP is decreased. These resultsare qualitatively similar to the phenomenon of uncompetitive inhibitionwhich may be ascribed to the presence of independent and separatebinding sites as well as a ping-pong binding mechanism (Dixon et al.,1979, Enzymes, 3rd Ed., Academic Press, New York).

[0217] Single-stranded DNA (ssDNA) was determined to be the most potentstimulator of hMSH2:hMSH6 heterodimer ATPase activity. Thus, theconflicting reports in the prior art regarding ATPase activities ofrelated MutS homologues may have resulted from contamination by ssDNAleached from columns used to purify the homologues and/or bynon-annealed ssDNA that remained following preparation ofoligonucleotide substrates.

[0218] hMSH2:hMSH6 Heterodimer Mismatch Binding is Abolished in thePresence of ATP in the Absence of Hydrolysis of ATP

[0219] Both bacterial and eukaryotic MutS homologs have been reported tofail to form a specific complex with a mismatched oligonucleotide in thepresence of ATP (Drummond et al., 1995, Science 268:1909-1912; Haber etal., 1991, EMBO. J. 10:2707-2715; Alani et al., 1997, Mol. Cell Biol.17: 2436-2447; Grilley et al., 1989, J. Biol. Chem. 264:1000-1004).Before the present invention, it was believed that ATP hydrolysiscatalyzed by MutS protein drove translocation of the protein along aduplex DNA strand, causing dissociation of the protein from any mismatchwith which it might be associated (Grilley et al., 1989, J. Biol. Chem.264:1000-1004; Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich,1991, Annu. Rev. Genet. 25:229-253; Modrich et al., 1996, Annu. Rev.Biochem. 65:101-133; Allen et al., 1997, EMBO J. 16:4467-4476). Thesuggestion that ATP hydrolysis was required for the mismatch release wasbased on the observation by others that adenylyl-imidodiphosphate(AMP-PNP), a non-hydrolyzable analog of ATP, does not alter mismatchbinding (Alani et al., 1997, Mol. Cell. Biol. 17: 2436-2447; Drummond etal., 1995, Science 268:1909-1912).

[0220] The experiments described in this Example establish that thehMSH2:hMSH6 heterodimer is released from a G/T-mismatched DNA substratein the presence of ATP, as indicated in FIGS. 2A and D. The value ofIC₅₀ (the concentration of ATP required to cause release of 50% of apopulation of heterodimers from a G/T-mismatched DNA substrate) wasdetermined to be approximately 3 micromolar.Adenosine-5′-O-3-thiotriphosphate (ATP-gamma-S), a poorly-hydrolyzableATP analog (Sekimizu et al., 1987, Cell 50:259-265; Yu et al., 1992, J.Mol. Biol. 225:193-216), caused a similar release of the hMSH2:hMSH6heterodimer from a G/T-mismatched DNA substrate, the value of IC₅₀ forATP-gamma-S being 3 micromolar, as indicated in FIGS. 2B and 2D.Addition of ADP to the mismatch binding reaction mixture resultedincreased binding affinity of the heterodimer for the G/T-mismatched DNAsubstrate, as indicated in FIGS. 2C and 2D.

[0221] The results presented in this Example demonstrate that release ofthe hMSH2:hMSH6 heterodimer from a G/T-mismatched DNA substrate withwhich it is associated is not dependent upon ATP hydrolysis. Thisconclusion follows from the observations that release of the complexoccurs in the absence of exogenous magnesium and that release of thecomplex from the substrate is effected by the presence of ATP-gamma-Sregardless of the presence or absence of magnesium. The presence ofmagnesium is absolutely required for hMSH2:hMSH6 heterodimer-dependentATP hydrolysis Furthermore, NS binding of hMSH2 to homoduplex DNA isinsensitive to the addition of exogenous ATP. Thus, the presence of ATPaffects only the ability of the hMSH2:hMSH6 heterodimer to bind tomismatched DNA substrates. Binding of the heterodimer to homoduplex DNAis not affected by ATP.

[0222] The presence of 2′-deoxy adenosine triphosphate (dATP) to themismatch binding reaction mixture caused release of a G/T-mismatched DNAsubstrate from the hMSH2:hMSH6 heterodimer, similarly to the releasecaused by the presence of ATP or ATP-gamma-S in the mixture, asillustrated in FIG. 3. No other nucleotide was found to stimulate therelease of the G/T-mismatched DNA substrate from the heterodimer.

[0223] Neither of two other non-hydrolyzable analogs of ATP, namelyAMP-PNP and adenyl-(beta-, gamma-methylene)-diphosphonate (AMP-PCP),caused release of the heterodimer from the substrate. Equilibriumcompetition between each of these two analogs and ATP suggested thatthey bind to the heterodimer and caused effects similar to those causedby ADP. Failure of AMP-PNP and AMP-PCP to stimulate release ofmismatched DNA from the heterodimer demonstrated that the interactionbetween the beta-gamma bridging oxygen atom of ATP and either theheterodimer or the mismatched DNA substrate bound to the heterodimer arefor release of the substrate from the heterodimer. Enzyme-nucleotidetriphosphate complexes in which the beta, gamma oxygen atom interactswith either the enzyme or its substrate are not unknown. For example,the Ras GTPase binds GTP, and donation of a hydrogen bond to thebeta-gamma bridging oxygen of GTP is thought to contribute to catalysisby the enzyme (Maegley et al., 1996, Proc. Natl. Acad. Sci. USA93:8160-8166).

[0224] The results presented in this example demonstrate that thehMSH2:hMSH6 heterodimer binds to a mismatched DNA substrate in thepresence of ADP, and that the substrate is released from the heterodimerin the presence of ATP or dATP. Because ATP-induced release of thesubstrate from the heterodimer does not require magnesium and issimilarly induced by ATP-gamma-S, ATP hydrolysis is not implicated insubstrate release. As increasing amounts of ATP or ATP-gamma-S wereadded to the mismatch binding reaction mixture, approximately 15% ofS-shifted material gradually became re-associated with the DNA in theform of a NS-shifted heterodimer, as indicated in FIGS. 2A and 2B. Thisfraction was consistent with the amount of NS binding observed forhomoduplex DNA at this concentration of the heterodimer, as indicated inFIG. 2B. These results indicated that hMSH2:hMSH6 heterodimers whichdissociated from mismatched substrate could re-associate with either theduplex arms or the ends of the substrate. ATP hydrolysis catalyzed bythe hMSH2:hMSH6 heterodimer results in recovery of mismatch bindingactivity of the heterodimer

[0225] To determine the role of ATP hydrolysis in mismatch recognition,ATP or ATP-gamma-S was introduced into a mismatch binding reactionmixture in the absence of magnesium. As illustrated in FIGS. 2A, 2B, 2D,and 3, introduction of either compound resulted in release of thehMSH2:hMSH6 heterodimer from the mismatched DNA substrate in the absenceof hydrolysis of the compound. In experiments presented in FIG. 4A,magnesium was added to each reaction mixture, which was maintained at37° C., and the G/T mismatch binding activity of hMSH2:hMSH6 heterodimerwas followed over time, with time zero corresponding to the time atwhich magnesium was added. In the reaction mixture comprising ATP,mismatched DNA substrate binding activity of the heterodimer wasinitially low, nearly 70% of this activity was recovered after tenminutes of incubation at 37° C., and more than 95% of the activity wasrecovered fifty minutes after magnesium addition. Substantially less(about 22%) of mismatched DNA substrate binding activity was recoveredin the reaction mixture to which ATP-gamma-S was added. These resultsdemonstrated that efficient hydrolysis by the heterodimer is essentialfor recovery of the heterodimer's mismatch binding activity.Substitution of ATP with DATP produced quantitatively similar recoveryof mismatch binding activity (i.e. >95% recovery) following incubationat 37° C. Taken together, these results demonstrated that the intrinsicATPase activity associated with the human hMSH2:hMSH6 heterodimer isrequired for recovery from mismatch-release induced by binding to and/orexchange with, ATP or dATP. [0199] Complete recovery of mismatched DNAsubstrate binding activity of the hMSH2:hMSH6 heterodimer, whichactivity was abolished by exposing the heterodimer to ATP, was achievedby increasing the ratio of the concentration of ADP to the ratio of ATPin the solution in which the heterodimer was suspended, as indicated inFIG. 4B In this competition experiment, mismatch binding reactionmixtures comprised 0.2 millimolar ATP, 1 millimolar MgCl₂, and aselected concentration of ADP from 0 to 3.2 millimolar. It wasdetermined that a 2- to 3-fold excess of ADP to ATP resulted in reversalof approximately half of the release of substrate by the heterodimercaused by the presence of ATP. Approximately complete reversal ofsubstrate release caused by the presence of ATP was achieved byproviding a 16-fold excess of ADP to the mixture. A qualitativelysimilar, though functionally opposite, result was obtained when thecompetition was performed by including a fixed concentration of ADP inthe reaction mixture and adding various concentrations of ATP. Thus, ADPand ATP are nearly equivalent in their ability to associate with thehMSH2:hMSH6 heterodimer, but the two nucleotides elicited oppositefunctional effects on mismatch binding. ATP caused release of substratebound to the heterodimer, and ADP induced binding of the substrate tothe heterodimer. Therefore, ADP is responsible for mismatch bindingrecovery.

[0226] Taken together, these observations support the conclusion thatthe hMSH2:hMSH6 heterodimer functions as a molecular switch, wherein theATP- (or dATP-) bound heterodimer is “OFF” (i.e. unable to associatewith a mismatched DNA substrate with which it is not already associated)and the ADP-bound heterodimer is “ON” (i.e. able to associate with amismatched DNA substrate with which it is not already associated). Amodel of the role of the hMSH2:hMSH6 heterodimer is illustrated in FIG.7.

[0227] ATP hydrolysis and ADP-ATP exchange determine mismatch bindingfunctions of the hMSH2:hMSH6 heterodimer

[0228] Steady-state analysis of an enzyme having ATPase activityreflects the rate-limiting step of the reaction, which can be eithergamma-phosphate hydrolysis or adenine nucleotide exchange. To understandthe mechanism of the ATPase activity exhibited by the hMSH2:hMSH6heterodimer and to further define the rate-limiting steps, bothhydrolysis and nucleotide exchange steps were directly examined.

[0229] Initial rate (i.e. single-turnover) analysis of an enzyme whichexhibits ATPase activity involves direct examination of the rate ofgamma-phosphate hydrolysis, and was performed using a method which issimilar to that used for the examination of regulators of G-proteinsignaling (RGS; Dohlman et al., 1997, J. Biol. Chem. 272:3871-3874). Inthese experiments, 0.2 micromolar [32P]gamma-ATP was contacted withhMSH2:hMSH6 heterodimer in the absence of magnesium, yielding aheterodimer having a [32P]gamma-ATP molecule bound thereto. At aselected time, magnesium and an excess of non-labeled ATP were added tothe reaction mixture, and the rate of a single-round of gamma-phosphatehydrolysis was assessed. Subsequent rounds of hydrolysis wereundetectable because the ATP hydrolyzed during those rounds was notlabeled. Because the calculated K_(cat) for ATP at 37° C. was in excessof 20 minute⁻¹, and because this rate was above the limit of detectionof this methodology, these initial rate experiments were performed at20° C. It was determined that the hMSH2:hMSH6 heterodimer rapidlyhydrolyzed ATP in either the presence or the absence of DNA. Theseresults indicated that gamma-phosphate hydrolysis was not the ratelimiting step in the steady-state ATP hydrolysis by the heterodimer.

[0230] The extent of ATP hydrolysis which could be detected wasequivalent to the total number of hMSH2:hMSH6 heterodimers which couldbe bound to ³²P-labeled ATP prior to the addition of magnesium. Themaximal extent of detectable ATP hydrolysis was determined to depend onthe amount of the G/T-mismatched DNA substrate present in the reactionmixture during binding of labeled ATP to the heterodimer, as indicatedin FIGS. 5A and 5B. When the concentration of the G/T-mismatched DNAsubstrate in to the reaction mixture exceeded the apparent K_(d) forG/T-mismatched DNA substrate (i.e. about 20 nanomolar), the maximalextent of ATP hydrolysis decreased, as indicated in FIG. 5B. Thisobservation indicated that binding of the hMSH2:hMSH6 heterodimer to amismatched DNA molecule prior to binding of ATP to the heterodimerinhibits binding of ATP to the mismatched DNA-bound heterodimer. Thisobservation is consistent with the pseudo-uncompetitive behavior deducedin the steady-state ATPase activity experiments described herein (Dixonet al., 1979, Enzymes, 3rd Ed., Academic Press, New York).

[0231] Adenine nucleotide exchange was assessed using a method similarto that used for guanine nucleotide exchange experiments involving Gproteins. In these studies, [3H]-ADP was contacted with hMSH2:hMSH6heterodimer in the presence of magnesium, yielding [³H]-ADP-boundheterodimer. At a selected time, an excess of non-labeled ATP was addedto the reaction mixture, and the amount of ADP that remained bound tothe heterodimer was assessed at selected times.

[0232] In the absence of DNA, incomplete ADP nucleotide exchange wasobserved during a 15 minute reaction period. The half-life of theADP-bound heterodimer was greater than eight hundred seconds. Theseresults clearly suggest that in the absence of DNA, replacement of ADPby ATP is the rate limiting step for the hMSH2:hMSH6 heterodimer ATPaseactivity.

[0233] In the presence of G/T-mismatched DNA substrate, nucleotideexchange was significantly more rapid, the half-life of the ADP-boundheterodimer being less than two seconds. Thus, it was demonstrated thatbinding of the heterodimer to a G/T-mismatched DNA substrate stimulatedreplacement of the labeled ADP molecule originally bound to theheterodimer by a non-labeled ATP molecule.

[0234] Taken together with the results obtained from the single turnoverhydrolysis experiments described herein, these observations indicatedthat in the absence of mismatched DNA, the hMSH2:hMSH6 heterodimer iscapable of a single ATP hydrolysis reaction that yields an ADP-boundheterodimer. While in the ADP-bound form, the heterodimer does notexchange ADP for ATP until the heterodimer binds to a DNA mismatch. Bybinding to a mismatch, the ADP-bound heterodimer becomes competent toexchange ADP for ATP. Exchange of ADP for ATP causes release of theheterodimer from the mismatch. ATP-bound heterodimer, when no longerbound to mismatched DNA, is capable of catalyzing ATP hydrolysis,yielding ADP-bound heterodimer, which is competent to bind to a DNAmismatch. These results indicate that the hMSH2:hMSH6 heterodimer is amolecular switch controlled by the phosphorylation state of the adeninenucleotide bound thereto.

[0235] Release of the hMSH2:hMSH6 heterodimer from a G/T-mismatched DNAsubstrate may occur by dissociation

[0236] Prior art models of mismatch recognition by MutS homologsimplicated ATP-dependent translocation and/or treadmilling along DNA asa mechanism for association and dissociation of the homolog with a DNAmismatch (Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich, 1991,Annu. Rev. Genet. 25:229-253; Modrich et al., 1996, Annu. Rev. Biochem.65:101-133; Allen et al., 1997, EMBO J. 16:4467-4476). Common to all ofthese prior art models is a postulated time-dependent unidimensionalhomolog displacement mechanism which occurs whether the homolog is boundto duplex DNA or mismatched DNA. In contrast, a simple dissociationmechanism would exhibit rapid and two-dimensional displacement of thehomolog from duplex DNA or mismatched DNA.

[0237] The ability to distinguish NS and S electrophoretic bandscorresponding to the homologous 81-base pair DNA substrate-boundhMSH2:hMSH6 heterodimer and the G/T-mismatched 81-base pair DNAsubstrate-bound heterodimer, as illustrated in FIG. 2A, provided anopportunity to examine the dissociation mechanism of the heterodimerfrom the G/T-mismatched DNA substrate, as well as from homoduplex DNA.In these experiments, the G/T-mismatched DNA substrate was bound to theheterodimer, and an excess of an unlabeled competitor DNA or an excessof ATP, or both, was added to the mixture. If a tracking or slidingmechanism of the prior art were operable for heterodimer dissociation,it would be expected that a time-dependent loss of the S shiftedelectrophoretic band of G/T-mismatched DNA substrate-bound complex wouldbe observed, and that a coincident gain of the NS electrophoretic bandwould be observed. If a simple dissociation mechanism were operable forheterodimer dissociation, it would be expected that loss of the Sshifted band would be observed without any coincident increase in theintensity of the NS shifted band because the vast excess of unlabeledhomoduplex DNA would preclude secondary reassociation of the complexwith the arms or ends of the labeled G/T-mismatched DNA substrate. Onepotential complication would be if the amount of time required forheterodimer enables diffusion of the dimer to a different position onthe DNA substrate were nearly the same as the time which would berequired for simple dissociation.

[0238] Three experiments were performed to determine the mechanism ofhMSH2/hMSH6 protein complex dissociation from a labeled 8 1-base pairG/T-mismatched DNA substrate. The results of these experiments areillustrated in FIG. 6.

[0239] In the first experiment, the stability of G/T-mismatched DNAsubstrate-bound hMSH2/hMSH6 complex was assessed by exposing themismatched substrate-bound complex to a 400-fold excess of non-labeledhomoduplex DNA and observing the intensities of S shifted and NS shiftedelectrophoretic bands at selected times, as illustrated in FIG. 6C.Examination of the gel depicted in FIG. 6C indicated that the S-shiftedelectrophoretic band, and thus the amount of the G/T-mismatched DNAsubstrate-bound hMSH2:hMSH6 heterodimer in the reaction mixture, was notreduced significantly over the ten minute incubation period. Thus, thehalf-life of the G/T-mismatched DNA substrate-bound hMSH2:hMSH6heterodimer was much greater than ten minutes, meaning that themismatched substrate-bound complex is stable in the presence of a vastexcess of homoduplex DNA.

[0240] In the second experiment, the stability of G/T-mismatched DNAsubstrate-bound hMSH2:hMSH6 heterodimer was assessed by exposing themismatched substrate-bound heterodimer to ATP and observing theintensities of S shifted and NS shifted electrophoretic bands atselected times, as illustrated in FIG. 6A. A gradual decrease in theintensity of the S shifted electrophoretic band was observed, the bandhaving a half life of about twenty seconds. Concurrently with thedecrease in the intensity of the S shifted electrophoretic band, agradual but not quantitative increase in the intensity of the NS-shiftedelectrophoretic band was observed. This observation indicated that ATPinduced a time-dependent reduction of specific binding of thehMSH2:hMSH6 heterodimer to the mismatched DNA substrate and that atleast a portion of the heterodimer reassociated with the mismatched DNAsubstrate in a non-specific manner. However, this experiment did notdistinguish between the tracking/sliding or simple dissociation andreassociation mechanisms.

[0241] In order to attempt to distinguish between translocation andsimple dissociation and reassociation, a third experiment was performed.In this experiment, the stability of G/T-mismatched DNA substrate-boundhMSH2:hMSH6 heterodimer was assessed by exposing the mismatchedsubstrate-bound heterodimer to both ATP and a 400-fold excess ofnon-labeled homoduplex DNA and observing the intensities of S shiftedand NS shifted electrophoretic bands at selected times (FIG. 6B). As inthe second experiment, a gradual decrease in the intensity of the Sshifted electrophoretic band was observed, the half-life of the bandagain being about twenty seconds. This observation was consistent withATP induction of dissociation of the heterodimer from the mismatched DNAsubstrate. However, under these conditions, no increase in the intensityof the NS electrophoretic band was observed. Together, theseobservations suggest that in the presence of excess non-labeledhomoduplex DNA, the dissociation of the heterodimer from mismatched DNAmight not proceed through the product corresponding to the NSelectrophoretic band, but instead may be instantaneous and irreversible.

[0242] When excess non-labeled homoduplex DNA was added to thehomologous 81-base pair DNA substrate, the NS electrophoretic bandassociated with the product formed by contacting the heterodimer withDNA substrate, as indicated in FIG. 1B, for example, could be detected,as indicated in FIG. 6D. This observation indicated that, even at 4° C.,the product corresponding to the NS band was exceedingly unstable andthat the level of hMSH2:hMSH6 heterodimer which remained associated withthe DNA substrate was less than the lower limit of accurate quantitationusing gel shift analysis.

[0243] The hMSH2:hMSH6 heterodimer acts as a molecular switch inmismatch recognition

[0244] The discovery that the hMSH2:hMSH6 heterodimer is a novelmolecular switch which is activatable by ADP was made by reconcilingnumerous observations described herein. These observations aresummarized as follows. ADP and ATP have opposing effects on the role ofthe hMSH2:hMSH6 heterodimer in mismatched DNA binding. Dissociation ofmismatched DNA from the hMSH2:hMSH6 heterodimer is not dependent uponATP hydrolysis. Hydrolysis of ATP by the hMSH2:hMSH6 heterodimer resultsin recovery of the ability of the heterodimer to associate withmismatched duplex DNA. gamma-Phosphate hydrolysis is not the ratelimiting step of ATPase activity catalyzed by the of the heterodimer.Displacement of ADP by ATP is the rate limiting step of ATPase activitycatalyzed by the hMSH2:hMSH6 heterodimer. Displacement of ADP from theof the heterodimer by ATP is accelerated in the presence of mismatchedduplex DNA, but hydrolysis of the gamma-phosphate bond is notaccelerated. ATP-dependent release of mismatched DNA from thehMSH2:hMSH6 heterodimer occurs rapidly, possibly by simple dissociationor by rapid ATP-hydrolysis-independent diffusion to a free end of theDNA. These observations indicate that gamma-phosphate hydrolysis anddisplacement of ADP by ATP determine whether the hMSH2:hMSH6 heterodimerbinds to or is released from mismatched duplex DNA, as illustrated inFIG. 7. Recognition of the hMSH2:hMSH6 heterodimer as a molecular switchsupports the conclusion that it is a trigger for determining the timingof subsequent excision repair-related events.

[0245] Implications for mismatch repair

[0246] The number of hMSH2:hMSH6 heterodimers in the nucleus of aproliferating cell has been estimated to exceed one thousand (Drummondet al., 1995, Science 268:1909-1912; Wilson et al., 1995, Cancer Res.55:5146-5150; Meyers et al., 1997, Cancer Res. 57:206-208). Thecalculated K_(d) of the heterodimer for mismatched DNA (i.e. about 20nanomolar) implies that a single mismatched nucleotide in a human cellis likely to be efficiently recognized and bound with high affinity byan hMSH2:hMSH6 heterodimer. In the presence of ATP, this high affinitybinding is nearly irreversible. Thus, dissociating the heterodimer frommismatched DNA in order to allow a subsequent excision repair event toproceed may be more difficult than binding the heterodimer to themismatch.

[0247] Generality of MutS function

[0248] The studies described in this Example, which involved the humanmismatch binding reaction catalyzed by the hMSH2:hMSH6 heterodimer, areconsistent with genetic studies performed in both bacteria and yeast. Inthose studies, mutation of the adenine nucleotide binding and hydrolysisdomain(s) resulted in a dominant mutator phenotype (Haber et al., 1991,EMBO. J. 10:2707-2715; Wu et al., 1994, J. Bacteriol. 176:5393-5400;Alani et al., 1997, Mol. Cell. Biol. 17: 2436-2447). Those studies,combined with the studies described in this Example, indicate that theremay be two opposing functional alterations of MutS homologs that cancause such a dominant mutator phenotype. First, alteration of theability of the homolog to bind and/or exchange ADP for ATP can cause adominant mutator phenotype. Second, alteration of the ability of thehomolog to hydrolyze ATP can similarly cause such a phenotype. Inabilityof the homolog to bind to ADP or to exchange ADP for ATP would result ina permanently mismatched DNA-bound form of the MutS homolog. This formof the homolog would exclude the repair machinery from the mismatchsite. Inability of the MutS homolog to hydrolyze ATP would result in aform of the homolog that would be unable to bind to mismatched DNA andwhich, therefore, would be unable to recruit the cellular mismatchrepair proteins and factors to the site of the mismatch. Each theseconditions would cause an increased mutation rate in the organismcontaining the homolog, as a consequence of the organism's depressedability to repair mismatched DNA (Wu et al., 1994, J. Bacteriol.176:5393-5400).

[0249] Preliminary studies performed using the methods described hereinand using purified Escherichia coli MutS protein suggest that E. coliMutS also functions as a molecular switch, albeit with a more stringentrequirement for mismatch-induced nucleotide exchange. Therefore, theproperties of the MutS homologs hMSH2 and hMSH6, as described hereinappear to be properties of all MutS homologs, including, but not limitedto, E. coli MutS, and the human MutS homologs hMSH2, hMSH3, and hMSH6.

[0250] Similarity of the hMSH2:hMSH6 heterodimer to G-protein switches

[0251] The hMSH2:hMSH6 molecular switch is, in some respects, similar toG-protein switches which have been described (Bokoch et al., 1993, FASEBJ. 7:750-759). G-proteins are known to trigger translocation eventsassociated with protein synthesis (Laalami et al., 1996, Biochimie78:577-589; Parmeggiani et al., 1981, Mol. Cell. Biochem. 35:129-158),cascade events associated with cell signaling (Medema et al., 1993,Crit. Rev. Oncol. 4:615-661; Wiesmuller et al., 1994, Cell Signal.6:247-267) and physiological responses to ligand-binding by membranereceptors (Spiegel, 1987, Mol. Cell. Endocrinol. 49:1-16). ManyG-proteins are associated with regulators that stimulate both the GTPaseactivity of the G-protein (Tocque et al., 1997, Cell Signal. 9:153-158)and the exchange of G-protein-bound GDP for GTP (Dohlman et al., 1997,J. Biol. Chem. 2 72:3871-3874; Quilliam et al., 1995, Bioessays17:395-404). In fact, the Ras G-protein was determined to be unable tocatalyze GTP hydrolysis because it is unable to exchange GDP for GTP.The discovery of a GTPase activating protein (GAP) that stimulated GTPgamma-phosphate hydrolysis, and a guanine nucleotide exchange factor(GNEF) that stimulated the exchange of GDP for GTP, provided a model forregulation of the Ras G-protein switch (Tocque et al., 1997, CellSignal. 9:153-158; Dohlman et al., 1997, J. Biol. Chem. 2 72:3871-3874).

[0252] It has therefore been discovered that protein regulation of theexcision-resynthesis processes associated with mismatch repair occurs bystimulation of the ATPase activity of the hMSH2:hMSH6 heterodimer or ofthe ability of the heterodimer to exchange ADP for ATP. The latterstimulation can occur either by stabilizing the ADP-bound form of theheterodimer or by stimulating exchange of ADP for ATP to effect releaseof the heterodimer from mismatched DNA. It is thought by the inventorsthat MutL homologs, such as the human MutL homologs, hMLH 1, hPMS 1, andhPMS2, perform these regulatory functions.

Example 2

[0253] A Mouse Construct Nullizygous for both msh2 and p53 and Methodsof Making and Use Thereof

[0254] Transgenic mice which are nullizygous for both Msh2 and p53 havebeen made, and are referred to herein as Msh2^(−/−)p53^(−/−) mice. Othertransgenic animals which are nullizygous for both Msh2 and p53, andwhich particularly include mammals, especially including rodents such asmice and rats, may be made using methods analogous to those describedherein and are useful in the screening methods described herein.

[0255] The development of female Msh2^(−/−)p53^(−/−) mouse embryos isphenotypically arrested at approximately the 9.5 day stage, andapoptosis is induced shortly thereafter in the cells of these embryos.Male Msh2^(−/−)p53^(−/−) mouse embryos are viable, but succumb to tumorssignificantly earlier than either Msh2^(−/−)p53^(+/±) orMsh2^(+/±)p53^(−/−) littermates (i.e. nullizygous Msh2 mice ornullizygous p53 mice, respectively). Furthermore, the frequency ofmicrosatellite instability (MSI) in tumor tissue obtained fromMsh2^(−/−)p53^(−/−) mice is not significantly different than thefrequency in tumor tissue obtained from Msh2^(−/−)p53^(−/−) mice.Synergism in tumorigenesis and independent segregation of the MSIphenotype suggest that Msh2 and p53 are not genetically epistatic.Msh2^(−/−)p53^(−/−) mice are useful as models of disease or disorderstates which cannot be identified in mice nullizygous for only one ofMsh2 or p53. Furthermore, Msh2^(−/−)p53^(−/−) mice are useful foridentifying compositions which affect the onset or progression of such adisease or disorder state. Thus, a Msh2^(−/−)p53^(−/−) mouse isparticularly useful as a model system for studying multisteptumorigenesis, apoptosis, and aging.

[0256] The materials and methods used in the experiments presented inthis Example are now described.

[0257] Generation of Msh2^(−/−)p53^(−/−) Mice

[0258] Methods for making heterozygous and nullizygous Msh2 mice andheterozygous and nullizygous p53 mice have been described (de Wind etal., 1995, Cell 82:321-330; Reitmair et al., 1995, Nature Genet.11:64-70; Donehower et al., 1992, Nature 356:215-221; Jacks et al.,1994, Curr. Biol. 4:1-7; Purdie et al., 1994, Oncogene 9:603-609).

[0259] Mice heterozygous for Msh2 (i.e. Msh2^(+/−)p53^(+/+) mice) on amixed C57BL/6J and 129/Ola background and mice heterozygous for p53(i.e. Msh2^(+/+p)53^(+/−) mice) on a mixed C57BL/6J and 129/Sv weremated to produce F1 progeny heterozygous for both genes (i.e.Msh2^(+/−p)53^(+/−) mice). Heterozygous sibling F1 progeny wereintercrossed to produce progeny nullizygous for both Msh2 and p53 (i.e.Msh2^(−/−)p53^(−/−) mice). Mice were genotyped using Msh2- and p53-specific PCR-based assays, using methods well known in the art.

[0260] Isolation of Genomic DNA

[0261] Mouse genomic DNA was extracted from ear-notched tissue of miceand from amniotic tissue of mouse embryos at 9.5, 11.5, or 13.5 days ofdevelopment, using a QIAamp Tissue Kit (Qiagen, Chatsworth, Calif.)according to the manufacturer's instructions.

[0262] PCR-based Genotyping of Mice

[0263] A three-primer assay specific for Msh2 was carried out asdescribed (Reitmair et al., 1995, Nature Genet. 11:64-70). A four-primerassay specific for p53 was carried out using 50 nanograms of templateDNA in a 50 microliter reaction mixture containing 1 unit of Taqpolymerase (Fisher Scientific, Malvern, Pa.) and 100 millimolar each ofthe following primers, each of which is identified with a five digitnumber and the sequence of each of which is listed:

[0264] 10681 (5′-GTGTTTCATT AGTTCCCCAC CTTGAC-3′; SEQ ID NO: 7);

[0265] 10480 (5′-ATGGGAGGCT GCCAGTCCTA ACCC-3′; SEQ ID NO: 8);

[0266] 10588 (5′-GTGGGAGGGA CAAAAGTTCG AGGCC-3′; SEQ ID NO: 9); and

[0267] 10930 (5′-TTTACGGAGC CCTGGCGCTC GATGT-3′; SEQ ID NO: 10).

[0268] The amplification reaction involved 35 cycles of amplification(94° C., 15 seconds; 56° C., 30 seconds; 72° C., 1 minute) using aPerkin-Elmer GeneAmp 9600 thermal cycler. The wild-type primers, 10681and 10480, amplified a product of about 320 base pairs length, and thetargeted allele (i.e. p53) primers, 10588 and 10930, amplified a productof about 150 base pairs length.

[0269] The gender of embryos was determined using primers specific forthe Y-chromosome gene as described (Sah et al., 1995, Nature Genet.10:175-180). The presence of the X-chromosome was confirmed separatelyin all cases using the following two X-chromosome specific primers toamplify the locus DXMIT6:

[0270] 5′-ACCATTCAAATTGGCAAGG-3′(SEQ ID NO: 11); and

[0271] 5′-GTGGCTCGAGTTGTTTGCAG-3′(SEQ ID NO: 12).

[0272] PCR cycling conditions were as described above for p53genotyping, except that the annealing temperature was 53° C., ratherthan 56° C. The X-chromosome specific primers amplified a product ofabout 210 base pairs in length. All PCR amplification products wereresolved by electrophoresis on a 2% (w/v) agarose gel alongside a 100base pair polynucleotide ladder standard and were visualized by ethidiumbromide staining.

[0273] Timed Pregnancies

[0274] Male and female mice having a known Msh2+/−p53^(+/−) Msh2^(+/−)p53^(−/−), or Msh2^(−/−) p53^(+/−) genotype were mated and each of thefemales was examined daily for the presence of a vaginal plug (anindicator of pregnancy which appears at about day 0.5 of embryodevelopment). Pregnant females were sacrificed at 13.5 days, at 11.5days, or at 9.5 days gestation. Embryos were dissected out from thepregnant females into Hank's Balanced Salt Solution (Gibco BRL, GrandIsland, N.Y.) under a dissecting microscope, fixed in 4% (v/v) bufferedformalin, and documented by photomicrography. Amnion was retrieved fromeach embryo, DNA was extracted therefrom, and the sex and genotype ofeach embryo was determined by PCR.

[0275] Histology

[0276] Tissue specimens were fixed in 10% (v/v) or 4% (v/v) bufferedformalin and embedded in paraffin. Histological analysis was carried outon 3 micrometer-thick sections stained with hematoxylin and eosin (H&E).

[0277] TUNEL Assay

[0278] Paraffin-embedded tissue sections were de-waxed and rehydratedusing a graded alcohol series, using methods well known in the art.Apoptotic cells and appropriate positive and negative control sampleswere analyzed using the In Situ Cell Detection Kit, AP with NBT/BCIP,manufactured by Boehringer Mannheim (Indianapolis, Ind.), according tothe manufacturer's instructions. TUNEL-stained tissue sections wereanalyzed both by fluorescence microscopy and light microscopy.

[0279] Kaplan-Meier Survival

[0280] Kaplan-Meier survival probability was calculated for mice thatwere found dead or were sacrificed when found to be moribund. The age ofthe mice was calculated in days. Because no mice died in the controlgroup, confidence limits could not be determined.

[0281] Microsatellite Instability in Lymphoid Tumors

[0282] Paired ear-notch (i.e. normal) and lymphoid tumor tissues wereanalyzed for microsatellite instability at five chromosomal loci:D17Mit123, D10Mit2, D6Mit59, D4Mit27, and D3Mit203. Microsatelliteprimer sequence pairs appropriate for amplification of these loci wereobtained from the World Wide Web site of the Whitehead Institute forGenome Research (http://www.genome.wi.mit.edu), and were chosen toamplify fragments containing at least twenty dinucleotide repeatsequences. PCR amplifications were carried out in a total reactionvolume of 25 microliters, using 50 nanograms of DNA as template, 100millimolar of each primer pair and 1 unit of Taq polymerase (FisherScientific, Malvern, Pa.). The amplification reaction involved 35 cyclesof amplification (94° C., 15 seconds; 56° C., 30 seconds; 72° C.minute). Amplified products were resolved by electrophoresis on a 6.7%(w/v) denaturing polyacrylamide gel and were visualized by silvernitrate staining of the gel.

[0283] The results of the experiments presented in this Example are nowdescribed.

[0284] Twenty-one Msh2^(−/−)p53^(−/−) mice were generated fromMsh2^(+/−)p53^(+/−), Msh2^(−/−) p53^(+/−), or Msh2^(+/−p)53^(−/−)parents. When the gender of each of the twenty-one Msh2^(−/−)p53^(−/−)mice was examined, all were determined to be male Msh2^(−/−)p53^(−/−)mice. The absence of female Msh2^(−/−)p53^(−/−) offspring is highlysignificant (p <0.001) and is unlikely to reflect the intrinsic bias formales observed in the colony from which the mice were derived, whereinthe normal male:female ratio is 181:138.

[0285] The fertility of male Msh2^(−/−)p53^(−/−) mice could not bedetermined, because they succumbed to tumors before they successfullymated. However, pathological examination of the testes of the maleMsh2^(−/−)p53^(−/−) mice did not reveal gross abnormalities uponautopsy, and histology revealed mature spermatogenesis in all twenty-oneof the male Msh2^(−/−)p53^(−/−) mice. Taken together, these resultssuggest that Msh2^(−/−)p53^(−/−) male mice are not sterile.

[0286] No gross morphological abnormalities were observed in Msh2^(−/−)animals either in utero or post-natally (de Wind et al., 1995, Cell82:321-330; Reitmair et al., 1995, Nature Genet. 11:64-70). In addition,the number of male and female Msh2^(−/−) mice in the studies describedherein was in accord with the expected 1:1 ratio, which suggests thatmale and female nullizygous Msh2 mice are equally viable. However, adecrease in the number of live born nullizygous p53 mice from theexpected Mendelian ratio was observed, which is qualitatively similar toprevious reports, although our limited numbers did not indicate a sexbias (Sah et al., 1995, Nature Genet. 10:175-180; Nicols et al., 1995,Nature Genet. 10:181-187).

[0287] No female Msh2^(−/−)p53^(−/−) mice were observed at weaning andnone of thirteen one-day-old pups which were found dead in the littersof mating pairs were Msh2^(−/−)p53^(−/−) Thus, all female embryosnullizygous for both Msh2 and p53 died in utero. To determine the pointin embryonic development at which these embryos died, numerous timedpregnancies were established. Because Msh2^(−/−)p53^(−/−) males were notavailable and Msh2−/−p53^(−/−) females were not viable, pairs of mice,each of which mice was a known Msh2^(+/−)p53^(+/−) Msh2^(+/−)p53^(−/−)Msh2^(−/−p)53^(+/−) mouse, were mated to produce Msh2^(−/−)p53^(−/−)embryos. Pregnant females were sacrificed at 9.5, 11.5, and 13.5 days ofgestation, the embryos were pathologically assessed for developmentaldefects and the genotype and gender of each embryo were determined byPCR. The results of these analyses are presented in Table 1. A total oftwenty-one embryos and six resorption sites were recovered from threefemales at day 13.5 of gestation. Of the twenty-one 13.5 day embryos,two male Msh2^(−/−)p53^(−/−) embryos and no female Msh2^(−/−)p53^(−/−)embryos were recovered, although a total of five Msh2^(−/−)p53^(−/−)embryos were statistically expected. Two 13.5 day embryos (one maleMsh2^(+/−)p53^(−/−) one female Msh2^(−/−)p53^(+/−) displayedexencephaly, while all other 13.5 day embryos appeared normal (Sah etal., 1995, Nature Genet. 10:175-180). TABLE 1 Sex and MorphologicalPhenotype of Timed Post-Implantation Embryos Female Male Days Resorption# of Embryos Msh^(-/-)p53^(-/-) Msh2^(-/-)p53^(-/-) Development SitesEmbryos Typed Nor Abnr Nor Abnr  e9.5 3 30 28 3 1 2 1 e11.5 11 21 17 0 42 0 e13.5 6 21 21 0 0 2 0 *28 — *96  *96  *0  *0  *21  *0 

[0288] In Table 1, embryos that arrested in development, that were inresorption, or that displayed gross abnormalities were classified asabnormal (Abnr), while those embryos which were not arrested indevelopment, were not in resorption, and did not display grossabnormalities were classified as normal (Nor). Thirteen newborn pupsthat were found dead, none of which were Msh2^(−/−)p53^(−/−) are notrepresented in this Table.

[0289] Twenty-one embryos and eleven resorption sites were recoveredfrom three pregnant females at day 11.5 of gestation. Of these, completePCR typing results were determined for seventeen embryos and oneresorption site. Five embryos were determined to be Msh2^(−/−)p53^(−/−)although eight Msh2^(−/−)p53^(−/−) embryos were statistically expected.Two of the five embryos were males that appeared morphologically normal(one is depicted in FIG. 9A), and three of the five embryos werefemales, all three of which had undergone developmental arrest, and allthree of which are depicted in FIGS. 9B, 9C, and 9D. The three femaleMsh2^(−/−)p53^(−/−) embryos appeared opaque and somites were notvisible. Based on the gross morphology of the three femaleMsh2^(−/−)p53^(−/−) embryos, it was estimated that they died at 9.5 daysof development. The tissue from the resorption site was typed as femaleMsh2^(−/−)p53^(−/−).

[0290] Thirty embryos and three resorption sites were recovered frompregnant females at day 9.5 of gestation. Twenty-eight embryos and oneresorption site were successfully typed. Two embryos and a resorptionsite were found to be male Msh2^(−/−)p53^(−/−), and four embryos weretyped as female Msh2^(−/−)p53^(−/−). Six Msh2^(−/−)p53^(−/−) embryoswere statistically expected. Neither of the male Msh2^(−/−)p53^(−/−)embryos exhibited any gross morphological abnormality. It is likely thatthe male Msh2^(−/−)p53^(−/−) resorption site represents a spontaneousabortion event. In one of the four female Msh2^(−/−)p53^(−/−) embryos,the anterior neural tube was not closed and the heart was not seen tobeat, which should occur around day 9 of development. These observationsare consistent with a developmental delay that could result from latefertilization or implantation or alternatively, from a developmentalabnormality that is apparent at day 9.5.

[0291] Paraffin embedded tissue sections from wildtype andMsh2^(−/−)p53^(−/−) female embryos, as depicted in FIG. 10, fromMsh2^(−/−) embryos, and from p53^(−/−) embryos were examined at day 11.5and at day 13.5. While the wildtype, Msh2^(−/−) and p53^(−/−) embryoshad clearly distinguished developmental features at day 11.5, thearrested Msh2^(−/−)p53^(−/−) female embryos contained noncohesive cellswithout preservation of embryonal tissue structures. In addition, H&Estained Msh2^(−/−)p53^(−/−) female embryonic tissue sections appeared tocontain an large number of “blebbed” structures typical of apoptoticcells. Furthermore, loss of nuclear hematoxylin stain typical fornecrosis was not observed in H&E stained Msh2^(−/−)p53^(−/−) femaleembryonic tissue sections (FIG. 10, Panel B).

[0292] TUNEL staining was performed on the paraffin embedded tissuesections (FIG. 10, Panels C-F). Although wildtype (FIG. 10, Panels C andE), Msh2^(−/−), and p53^(−/−) embryos displayed circumscribed foci ofapoptotic cells characteristic of normal embryonal development,Msh2^(−/−)p53^(−/−) female embryos displayed global catastrophicapoptosis (FIG. 10, Panels D and F). Furthermore, fluorescence TUNELstaining of Msh2^(−/−)p53^(−/−) female embryos revealed a speckledintracellular patterning characteristic of fragmented chromatin (FIG.10, Panel F). It was estimated that between about 60% and about 90% ofcells in Msh2^(−/−)p53^(−/−) female embryos were undergoing visibleapoptosis, as assessed by H&E and TUNEL staining.

[0293] Kaplan-Meier survival analysis was performed on a cohort ofninety-six mice, the data for which analysis are graphically presentedin FIG. 11. Msh2^(−/−)p53^(−/−) mice began to die of generalizedlymphomas at day 53 after birth and all twenty-one Msh2^(−/−)p53^(−/−)mice were dead within four months of birth. In contrast, only 18% (eightof forty-four) of Msh2^(−/−) littermates and 71% (five of seven) ofp53^(−/−) littermates were dead at the time the mice were analyzed.Thus, Msh2^(−/−)p53^(−/−) mice had a significantly (p<0.001) reducedmedian survival time of 73 days compared with the median survival timeof either Msh2^(−/−) mice (i.e. 200 days) or p53^(−/−) mice (i.e. 149days). Furthermore, all twenty-four wild-type (i.e. Msh2^(+/±)p53^(+/±))littermates were alive after approximately ten months. These resultsindicate that Msh2 and p53 null mutations cooperatively promotetunorigenesis. p53 has also been shown to cooperate with a variety ofother genes in mouse tumorigenesis models (Blyth et al., 1995, Oncogene10:1717-1723; Williams et al., 1994, Cold Spring Harbor Symp. Quant.Biol. 59:449-457; Williams et al., 1994, Cell 79:329-339; Donehower etal., 1995, Genes Dev. 9:882-895; Nacht et al., 1996, Genes Dev.10:2055-2066). However, as is apparent from FIG. 11, the effect ontumor-related death of having dual null mutations of Msh2 and p53 isgreater than the sum of the effects of having a single null mutation inMsh2 or p53 alone. Thus, the Msh2^(−/−)p53^(−/−) mouse described hereinhas a phenotype which is significantly different from a mere combinationof the phenotype of a Msh2^(−/−) mouse and the phenotype of a p53^(−/−)mouse.

[0294] Pathological examination of tumors showed that all twenty-oneMsh2^(−/−)p53^(−/−) mice developed highly aggressive generalizedlymphomas involving major organs. In addition, a pleomorphic sarcoma inthe flank, a malignant fibrous histiocytoma of the neck, and a tubularadenoma of the small intestine were observed, while other epithelialneoplasms were not detected. The tumor spectrum of Msh2^(−/−) andp53^(−/−) mice appeared similar to previous observations (de Wind etal., 1995, Cell 82:321-330; Reitmair et al., 1995, Nature Genet.11:64-70; Donehower et al., 1992, Nature 356:215-221; Jacks et al.,1994, Curr. Biol. 4:1-7; Purdie et al., 1994, Oncogene 9:603-609). Thetumor spectrum of Msh2^(−/−)p53^(−/−) mice differs significantly fromthe tumor spectrum of either Msh2^(−/−) mice or p₅₃ ^(−/−) mice. Thus,Msh2^(−/−) p53^(−/−) mice have utility different from that of eitherMsh2^(−/−) mice or p53^(−/−) mice.

[0295] Normal and tumor tissues obtained from individualMsh2^(−/−)p53^(−/−) mice were examined for microsatellite instability atfive loci: D17Mit123, D10Mit2, D6Mit59, D4Mit27, and D3Mit203. Theresults of these MSI studies are presented in Table 2. The frequency ofMSI in tumor tissues obtained from Msh2^(−/−) mice was not significantlydifferent (p>0.05) from the frequency of MSI in tumor tissues obtainedfrom Msh2^(−/−)p53^(−/−) mice. Microsatellite instability was notobserved in lymphomatous tumor tissue obtained from the seven p53^(−/−)mice examined. The observation that Msh2^(−/−)p53^(−/−) mice developedearlier onset of tumor-related disease, combined with the observedseparate segregation of the MSI phenotype with the Msh2 allele, suggeststhat Msh2 and p53 are not genetically epistatic. TABLE 2 The Frequencyof Microsatellite Instability in p53^(−/−), Msh2^(−/−), andMsh2^(−/−)p53^(−/−)Mice Tumors Genotype Tumor/ Examined MSI at >1 MSIat >2 MSI at >3 Normal Pairs (n) Locus Loci Loci p53^(−/−) 7 0 (0%) 0(0%) 0 (0%) Msh2^(−/−) 8 6 (75%) 4 (50%) 3 (38%) *Msh2^(−/−)p53^(−/−) 2117 (81%) 14 (67%) 12 (57%)

[0296] It is remarkable that female Msh2^(−/−)p53^(−/−) mouse embryosunderwent global developmental arrest and that widespread apoptosis ofthe cells of such embryos occurred around day 9.5 of development. Thatthese embryos underwent implantation and gastrulation strongly suggeststhat they are capable of executing the earlier stages of embryogenesis.The arrested phenotype is reminiscent of that described for a smallproportion of female p₅₃ ^(−/−) 0 mice (Sah et al., 1995, Nature Genet.10:175-180). However, unlike p53^(−/−) mice, no normal femaleMsh2^(−/−)p53^(−/−) mice or embryos were observed beyond 9.5 days ofembryonic development. This observation supports the conclusion that thefemale embryonic lethality of Msh2^(−/−)p53^(−/−) mice is highlypenetrant. In addition, none of the female Msh2^(−/−)p53^(−/−) embryosdisplayed the exencephaly that characterized the p53^(−/−) mice (Sah etal., 1995, Nature Genet. 10:175-180). Furthermore, while there was nodifference in apoptosis observed in developing p53^(−/−) mouse embryos,global catastrophic apoptosis was clearly observed in all theMsh2^(−/−)p53^(−/−) female mouse embryos examined at day 9.5 ofdevelopment. These results suggest that female Msh2^(−/−)p53^(−/−) micesuccumb at an earlier stage and by an entirely different pathology thanp53^(−/−) mice.

[0297] Without being bound to any particular theory, the lethalityobserved in female Msh2^(−/−)p53^(−/−) mouse embryos is consistent withthe following explanation. In the female embryonic lineage, dosagecompensation is achieved by random X chromosome inactivation around thetime of gastrulation, at which time intense embryonic cellularproliferation and apoptosis promote embryonic differentiation (Lyon,1961, Nature 190:372-373; Rastan, 1994, Curr. Opin. Genet. Dev.4:292-297; Theiler, 1972, In: The House Mouse Developement and NormalStages from Fertilization to 4 Weeks of Age, Springer-Verlag, New York,p. 168). The global apoptotic effect need not occur coincidentally withX chromosome inactivation. The full effect of dysregulation may onlybecome apparent after a number of cell divisions when the embryoundergoes a further burst of proliferation during embryonic ‘turning’between 8 and 9.5 days.

[0298] It has been shown that the inactivated X chromosome replicateslate in S phase (Taylor, 1960, J. Biophys. Biochem. Cytol. 7:455-464;Tagaki, 1974, Exp. Cell. Res. 86:127-1350. In addition, cells deficientin p53 have been shown to be defective for damage-induced G₁/Scheckpoint arrest, and cells that are deficient in MMR have been shownto be deficient for damage-induced G₂/M checkpoint arrest (Baker et al.,1990, Science 249:912-915; Diller et al., 1990, Mol. Cell. Biol.10:5772-5781; Lin et al., 1992, Proc. Natl. Acad. Sci. USA 89:9210-9214;Hawn et al., 1995, Cancer Res. 55:3721-3725; Marra et al., 1996,Oncogene 13:2189-2196). Thus, female-specific Msh2^(−/−)p53^(−/−) embryolethality may result from dysregulation of damage-induced arrestcheckpoint control, wherein such dysregulation is caused by a deficiencyof both p53 and Msh2, and whereby such dysregulation results in aninability of Msh2^(−/−)p53^(−/−) cells to arrest cell division andrepair damage introduced into the late replicating inactive Xchromosome. Such damage could take the form of non-replicated regions orchromosomal fragments that have resulted from inappropriate celldivision prior to the completion of inactive X chromosome replication.Fragmented, reactivated, or otherwise altered inactive X chromosomes maythen lead to global catastrophic cellular failure, developmental arrest,and apoptosis. Furthermore, the observation that the highest levels ofp53 mRNA are detected in wild-type embryos between 9 and 11 days ofdevelopment suggests an important role for p53 protein within this timeframe (Rogel et al., 1985, Mol. Cell. Biol. 5:2851-2855).

Example 3

[0299] A Discussion of hMSH2:hMSH6 Heterodimers in the Context ofMismatch Repair, Molecular Switches, and Signal Transduction

[0300] The foundation of molecular switches in biology is grounded intranslation elongation and cellular signal transduction. In thesesystems, guanine nucleotide-bound proteins (G-proteins) produce the ONand OFF signaling states that act as gates for downstream biochemicalprocesses. Experimental results described herein, in view of the resultsof studies by others, suggest that a similar molecular switch relies onadenine nucleotide-bound forms (A-proteins) to produce an ON and OFFsignaling state related to mismatched DNA repair and possibly to otherprocesses. In the field of signal transduction, the concept of amolecular switch is elementary, while the biochemical processes of DNArepair appear foreign. Similarly, the field of DNA repair recognizes thecomplex machinery required for DNA manipulation events, but regardsbiochemical signaling processes as essential cellular input which isoutside the genome juggernaut.

[0301] Genetics of Mismatch Repair

[0302] There are at least three ways in which mismatched nucleotidesarise in DNA. Physical or chemical damage to the DNA and its precursors,such as de-amination of 5-methyl-cytosine, can give rise to mismatchedbases (Friedberg, 1990, DNA Repair W.H. Freeman Co., New York).Misincorporation of nucleotides during DNA replication can yieldmismatched base pairs as well as the insertion and deletion ofnucleotides (for review see: Kolodner, 1996, Genes Dev. 10:1433-1442;Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich, 1997, J. Biol.Chem. 272:24727-24730). Genetic recombination produces regions ofheteroduplex DNA which may contain mismatched nucleotides when suchheteroduplexes result from the pairing of two different parental DNAsequences (Holliday, 1964, Genet. Res. 5:282-304). Mismatchednucleotides produced by each of these mechanisms are known to berepaired by enzyme systems that are both specific and overlapping(Friedberg, 1990, DNA Repair, W.H. Freeman Co., New York).

[0303] The most extensively studied system for mismatch repair (MMR) isthe DNA adenine methylation (Dam)-instructed pathway of Escherichia coli(Modrich, 1989, J. Biol. Chem. 264:6597-6600; Modrich and Lahue, 1996,Annu. Rev. Biochem. 65:101-133). The Dam-Instructed pathway promotes along-patch (approximately 2 kilobase pair) excision repair reactionwhich is genetically dependent on the mutH, mutL, mutS, and mutU (uvrD)gene products. Discrimination of the newly replicated DNA strand fromthe original template DNA strand is dependent on transientunder-methylation of the adenine nucleotide within GATC Dam sequences.The MutHLS pathway appears to be the most active MMR pathway in E. coliand is known to both increase the fidelity of DNA replication as well asto act on recombination intermediates containing mis-paired bases(Fishel et al., 1983, UCLA Symp. Mol. Cell. Biol. New Series 11:309-324;Fishel et al., 1986, J. Mol. Biol. 188:147-157).

[0304] Homologs of prokaryotic MutS and MutL proteins have beenidentified in nearly every organism with the exception of Archaea(Fishel et al., 1997, Curr. Opin. Genet. Dev. 7:105-113; Kolodner, 1996,Genes Dev. 10: 1433-1442). At present, there are 41 MutS homologs and 21MutL homologs in the NCBI database. In S. cerevisiae, six MutS homologs(MSH1-MSH6) and three MutL homologs (MLH1, MLH2, PMS1) have beenidentified. In human cells, a nearly identical set of five MutS homologs(hMSH2-hMSH6) and three MutL homologs (hMLH1, hPMS1, and hPMS2) areknown (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA 93:13629-13634;Bronner et al., 1994, Nature 368:258-261; Burns et al., 1994, Genes Dev.8:1087-1105; Fishel et al., 1993, Cell 75:1027-1038; Fujii et al., 1989,J. Biol. Chem. 264:10057-10064; Hollingsworth et al., 1995, Genes Dev.9:1728-1739; Kramer et al., 1989, J. Bacteriol. 171:5339-5346; Linton etal., 1989, Mol. Cell. Biol. 9:3058-3072; Mankovich et al., 1989, J.Bacteriol. 171:5325-5331; New et al., 1993, Mol. Gen. Genet. 239:97-108;Nicolaides et al., 1994, Nature 371:75-80; Palombo et al., 1995, Science268:19121-19914; Prolla et al., 1994, Mol. Cell. Biol. 14:407-415;Reenan et al., 1992, Genetics 132:963-973). Yet, with the exception ofgram-negative bacteria, there do not appear to be homologs of MutH.Thus, the mechanism of strand discrimination in even close relatives ofE. coli, the gram-positive bacteria, remains a mystery. The multipleMutS and MutL homologs have been found to participate in the diverseactivities of nuclear (MSH2, MSH3, MSH6, MLH1, PMS1) and organellar(MSH1) post-replication mismatch repair as well as having distinctmeiotic functions (MSH4, MSH5) (Fishel et al., 1997, Curr. Opin. Genet.Dev. 7:105-113; Kolodner, 1996, Genes Dev. 10: 1433-1442).

[0305] Biochemistry of Mismatch Repair

[0306] Purification and reconstitution studies by Modrich and colleagueshave led to a biochemical model for post-replication mismatch repair inE. coli. The reconstituted system requires the MutH, MutL, MutS and UvrD(helicase II) proteins along with DNA polymerase III holoenzyme, DNAligase, single-stranded DNA binding protein (SSB) and one of thesingle-stranded DNA exonucleases, Exol, ExoVII or RecJ (Cooper et al.,1993, J. Biol. Chem. 268:11823-11829; Grilley et al., 1989, J. Biol.Chem. 264:1000-1004; Lahue et al., 1989, Science 245:160-164; Lu et al.,1983, Proc. Natl. Acad. Sci. USA 80:4639-4643; Su et al., 1986, Proc.Natl. Acad. Sci. USA 83:5057-5061; Welsh et al., 1987, J. Biol. Chem.262:15624-15629). In this widely held biochemical model, initiation of aMMR event occurs when MutS recognizes and binds mis-paired nucleotidesthat result from polymerase misincorporation errors (Su et al., 1986,Proc. Natl. Acad. Sci. USA 83:5057-5061). It is suggested that MutSmismatch binding is followed by interaction with the MutL protein(Grilley et al., 1989, J. Biol. Chem. 264:1000-1004), which has beenproposed to accelerate an ATP-dependent translocation of the MutS-MutLcomplex (Allen et al., 1997, EMBO J. 16: 4467-4476) to a hemi-methylatedGATC Dam site bound by MutH (Welsh et al., 1987, J. Biol. Chem.262:15624-15629). The MutS-MutL complex then stimulates an intrinsicendonuclease activity of MutH which results in a specific strandscission on the non-methylated newly replicated DNA strand (Cooper etal., 1993, J. Biol. Chem. 268:11823-11829; Lahue et al., 1989, Science245:160-164; Welsh et al., 1987, J. Biol. Chem. 262:15624-15629). Thisstrand scission directs one of three single-stranded exonucleases (RecJ,Exo I, ExoVII) to degrade the newly replicated strand, which is thenre-synthesized by the Po1III holoenzyme complex (Lahue et al., 1989,Science 245:160-164). The net result is a strand-specific mismatchrepair event which can be bidirectional. Many of the genetic studiesperformed with this system appear to support this biochemicalinterpretation. For example, mutH, mutL, and mutS bacteria exhibit amutator phenotype that is presumed to be the result of the increasedprobability of misincorporation errors leading to mutations (Demerec etal., 1957, Carnegie Inst. Wash. Yearbook 370:390-406; Hill, 1970, Mutat.Res. 9:341-344; Miyake, 1960, Genetics 45:755-762; Siegel et al., 1967,J. Bacteriol. 94:38-47). However, not all predictions arising from thismodel agree with the genetic results. For example, recJ exol exoVIIbacteria do not appear to exhibit a mutator phenotype (Harris et al.,1998, J. Bacteriol. 180:989-993), suggesting that there may be otherexonuclease(s) or mechanism(s) involved in the mismatch repair process.

[0307] Functions for the Mismatch Repair Proteins

[0308] An activity exhibited by mismatch repair proteins is the specificmis-pair binding activity ascribed to MutS homologues (Acharya et al.,1996, Proc. Natl. Acad. Sci. USA 93:13629-13634; Chi et al., 1994, J.Biol. Chem., 269:29984-29992; Drummond et al., 1995, Science268:1909-1912; Fishel et al., 1994, Science 266:1403-1405; Gradia etal., 1997, Cell 91:995-1005; Marsischky et al., 1996, Genes Dev.10:407-420; Su et al., 1986, Proc. Natl. Acad. Sci. USA 83:5057-5061). Aclear function of the MutL homologs has, until the present invention,not been clear. Classification of MutS and MutL homologs is based on therecognition of highly conserved regions of amino acid identity. The mosthighly conserved region of the MutS homologs is confined to a region ofapproximately 150 amino acids that encompass a helix-turn-helix domainassociated with a Walker-A adenine-nucleotide and magnesium bindingmotif. Such motifs were described by Walker et al. (1982, EMBO J.1:945-951). This adenine nucleotide binding domain constitutes 100% ofthe identity between the known MutS homologs (Fishel et al., 1997, Curr.Opin. Genet. Dev. 7:105-113). Purified bacterial, yeast, and human MutShomologs exhibit an intrinsic low-level ATP hydrolytic (ATPase) activity(Alani et al., 1997, Mol. Cell. Biol. 17: 2436-2447; Chi et al., 1994,J. Biol. Chem., 269:29984-29992; Gradia et al., 1997, Cell 91:995-1005;Haber et al., 1991, EMBO J 10:2707-2715). This ATPase activity is likelyto be important for the function of the MutS homologs, as evidenced bythe observation that mutation of a conserved lysine residue in theadenine nucleotide binding domain results in a dominant mutatorphenotype in both bacteria and yeast (Alani et al., 1997, Mol. Cell.Biol. 17: 2436-2447; Haber et al., 1991, EMBO J. 10:2707-2715).

[0309] The most widely held model for MMR suggests MutS mis-pair bindingis followed by MutL association that results in an energy dependenttranslocation of this complex to a hemi-methylated Dam site occupied bythe MutH protein. In retrospect, this appears to have been a simplisticview since the rate of ATP hydrolysis (k_(cat)□10 min⁻¹) is unlikely tobe sufficient to drive mechanical translocation the, on average, severalhundred to thousand nucleotides required to encounter a MutH boundhemimethylated site. For example, if one ATP was required to translocateone nucleotide, as the most well accepted mechanism suggests, then itwould take 25-100 minutes to encounter a MutH on average. Yet,re-methylation of the transiently hemimethylated Dam sites has beenfound to occur within 0.1 to 3 minutes of passage of the replicationfork (Campbell et al., 1990, Cell 62:967-979). While the ATPase activitycould in theory be significantly faster in vivo, no stimulatory factorhas been identified to date in spite of an extensive search. Inaddition, the prevailing mechanism does not adequately account for MutLfunction nor the highly conserved domains recognized between MutLhomologs from bacteria to man (regions containing 100% identity in 21homologs).

[0310] The hMSH2-hMSH6 Molecular Switch

[0311] As described herein in Example 2 and elsewhere, human MutShomolog dimers, such as the hMSH2:hMSH6 heterodimer, function asmolecular switches responsible for the timing of mismatch repair, asillustrated in FIG. 7. This conclusion is based on the observationsthat:

[0312] 1) The ADP-bound heterodimer has high affinity for mismatchednucleotides;

[0313] 2) exchange of ADP for ATP results in release of the heterodimerfrom mismatched duplex DNA in the absence of hydrolysis;

[0314] 3) release of the heterodimer from mismatched duplex DNA occursby hydrolysis-independent diffusion off the ends of the shortoligonucleotides used in the experiments described in Example 2, asconfirmed by the experiments described in Example 4 herein; and

[0315] 4) hydrolysis of ATP results in recovery of the mismatch-bindingcompetent ADP-bound heterodimer.

[0316] The rate-limiting step and the ultimate control of thehMSH2:hMSH6 molecular switch is likely to be ADP to ATP exchange, whichis exceedingly inefficient in the absence of mismatched duplex DNA. Thecharacteristics of the hMSH2:hMSH6 heterodimer appear analogous to thecharacteristics of G-protein mediators of seven-transmembrane (7-TM)domain receptor signaling such as that used by the beta-Adrenergic andRhodopsin Receptors and the prototypical oncoprotein/G-protein Ras(Tocque et al., 1997 Cell Signal. 9:153-158). More specifically, theobservation that the hMSH2:hMSH6 heterodimer is induced to exchange ADPfor ATP in the presence of mismatched duplex DNA and then dissociatesfrom the mismatched portion of the duplex DNA to transduce a signal, isanalogous to the observation that ligand binding by 7-TM receptorsinduces associated G-proteins to exchange GDP to GTP and dissociate fromthe receptor to transduce a signal.

[0317] These similarities suggest two related models for mismatch repairthat are fundamentally different from all previously suggested models.These models are each based on the concept that MutS and its homologsare a novel type of molecular switch which determines the timing and/orappropriate assembly of repair components. The apparent affinity of thehMSH2:hMSH6 heterodimer for mismatched duplex DNA (Kd =about 2-20nanomolar) suggests that a single mismatch in a human cell should beefficiently recognized and bound. Furthermore, binding of thehMSH2:hMSH6 heterodimer to mismatched duplex DNA is slightly stabilizedin the presence of ADP. We would propose two non-exclusive models.

[0318] In the first model, tight binding of the ADP-bound form of thehMSH2:hMSH6 heterodimer to mismatched duplex DNA acts as a flag for theassembly or nearby localization of DNA excision repair components. Whenthe complete excision repair complex is assembled, exchange of ADP forATP is triggered and the hMSH2:hMSH6 heterodimer is released from themismatched portion of the duplex DNA, thus signaling exonucleolyticexcision and resynthesis of the region containing the mismatchednucleotide. Once released from the mismatched portion of the duplex DNA,the intrinsic ATPase activity of hMSH2-hMSH6 hydrolyzes bound ATP,resulting in a form that is once again competent for mis-pair binding.

[0319] In the second model, recognition of mismatched duplex DNA by theADP-bound form of the hMSH2:hMSH6 heterodimer provokes ADP to ATPnucleotide exchange. ATP-hydrolysis-independent DNA-associated diffusionof the hMSH2:hMSH6 heterodimer away from the mismatch portion of theduplex DNA to the assembled (or partially assembled) DNA mismatch repaircomplex. Activation of these components by the confederation of theATP-bound form of the hMSH2:hMSH6 heterodimer either engages the repairprocess (signaling the timing of mismatch repair as above) or triggersassembly of the remaining DNA mismatch repair components. Thisactivation event results in release of the hMSH2:hMSH6 heterodimer fromthe duplex DNA, hydrolysis of ATP bound to the hMSH2:hMSH6 heterodimer,and recycling of the form of the hMSH2:hMSH6 heterodimer capable ofassociating with mismatched duplex DNA. An advantage of this secondmodel is that the hMSH2:hMSH6 heterodimer remains associated with theDNA in an activated-form, poised to transduce the mismatch signal to anynearby mismatch repair components.

[0320] As a free protein complex, the hMSH2:hMSH6 heterodimer does notefficiently exchange ADP remaining after hydrolysis of ATP boundthereto, providing a long-term mismatch recognition-competent molecule.A key difference in the mismatch repair models described above and thosepreviously proposed, is the concept that ATP hydrolysis is not requiredto physically transduce the mismatch binding signal to downstream DNAmismatch repair components. Instead, ATP hydrolysis is required only torecycle the mis-pair recognition component (i.e. the hMSH2:hMSH6heterodimer). Without wishing to be bound by any particular theory, itis thought that the signal state of the hMSH2:hMSH6 heterodimer isrelated to the conformational state of the heterodimer, which in turn isrelated to whether ADP or ATP is bound thereto.

[0321] One of the most important observations concerning G-proteins istheir regulation by associated proteins (Bokoch et al., 1993, FASEB J.7: 750-759). There are two halves to the GTPase cycle: gamma-phosphatehydrolysis and GDP to GTP nucleotide exchange. Both of these steps canbe regulated either by inhibition or acceleration of these partialreactions. For example, the Ras protein has an remarkably sluggishintrinsic GTPase activity (Trahey et al., 1987, Mol. Cell. Biol.7:541-544), which can be accelerated at least 104-to 105-fold by aGTPase Activating Protein (GAP) (Trahey et al., 1987, Science238:542-545). In addition, there are other Regulators of G-ProteinSignaling (RGS) that singularly accelerate GTP gamma-phosphatehydrolysis, and GDP to GTP exchange stimulators (GES) and guaninedissociation inhibitors (GDI) that singularly affect nucleotide exchange(Dohlman et al., 1997, J. Biol. Chem. 272:3871-3874; Quilliam et al.,1995, Bioessays 17:395-404; Tocque et al., 1997, Cell Signal 9:153-158).It has been discovered herein that MutL homologs perform analogousfunctions (i.e. accelerate ATP gamma-phosphate hydrolysis, and ADP toATP exchange) with respect to MutS homologs.

[0322] Biological Switches and the Second Law of Thermodynamics

[0323] One could argue that the concept of a singular ON or OFF state ina molecular switch might violate the second law of thermodynamics. Thislaw requires that biochemical systems transit one state to the other bya series of microscopically reversible steps. This idea is based instatistical mechanics as it is applied to a system at equilibrium—whichmust be applied a priori to enzyme catalyzed biological processes. It iseasy to visualize the origins of the principle of microscopicreversibility by considering the consequences were it NOT true. Forexample, if the rate of A to B were greater than B to A at equilibrium,each of the rates B to C, C to D, and D to A would also have to begreater than their reverse rates in order to prevent build-up of theconcentration of any species, which is not permitted at equilibrium. Inthis case there would be a preferred direction-of-operation of thereaction cycle. Such a spontaneous cycle in a system at equilibrium(i.e. an engine that spontaneously produces work) is not consistent withthe drive toward maximum entropy contained in the second law ofthermodynamics.

[0324] There is no violation of the second law of thermodynamics if thetransit from an OFF to ON state (or visa versa) occurs reversibly. Themolecular basis for this type of microscopic reversibility can bevisualized for the MutS dimer and G-protein switches as reversiblenucleotide-binding as well as intermediate protein conformationalchanges that occur while transiting the extreme states. It is theseconformational transitions that determine interaction with effectorswhich is ultimately accounted for by the hydrolysis of NTP. Moresignificantly, one can experimentally affect the equilibrium of eachstate by altering the ratio of NDP/NTP in the absence of any hydrolysis,as indicated in FIG. 4B. It is also important to note that microscopicreversibility has been directly demonstrated for the “gated” maxi K⁺ionpump, which is a molecular switch controlled by similar conformationaltransitions (Song et al., 1994, Biophys. J. 67:91-104). Thus, molecularswitches are both reversible and, at equilibrium, clearly preserving afundamental tenant of thermodynamics.

[0325] Similarities Between Signal Transduction and DNA Metabolism

[0326] The use of controlled molecular switches appears to pervade allaspects of biology. From the standpoint of DNA metabolism, switchcontrolled processes appear mechanistically sensible. It is well knownthat the cellular components which perform replication, recombination,repair, and chromosome segregation are very large and composed ofmultiple subunits (Alberts, 1998, Cell 92:291-294). Analogous to anassembly-line for an automobile or an airplane, the assembly of DNAmetabolic machines must be done precisely and in a specific order toensure appropriate function. A series of well defined switches couldlogically control the progression of such an ordered assembly process.

[0327] The same type of switch-controlled cascade events that transducecellular signals may also control DNA metabolic events. An importantdifference between these switches is the identity of the nucleotide thatinduces the conformational transitions associated with signaling. At themoment the general rule seems to be that guanine nucleotides areinvolved in cellular signaling events and adenine nucleotides areinvolved in DNA metabolic signaling events.

Example 4

[0328] Interactions of hMSH2 with hMSH3 and of hMSH2 with hMSH6:Examination of Mutations Associated with HNPCC

[0329] In the experiments described in this Example, mutations in thehuman mismatch repair protein hMSH2 were determined to co-segregate withthe occurrence in individuals afflicted with hereditary non-polyposiscolorectal cancer (HNPCC). As described herein, hMSH2 forms specificmis-pair binding complexes with hMSH3 and hMSH6. These proteininteractions were further characterized by mapping the contact regionsbetween the monomers of the hMSH2:hMSH3 and hMSH2:hMSH6 heterodimers.

[0330] The results described in this Example demonstrate that there areat least two distinct regions of monomer:monomer interaction in bothhMSH2:hMSH3 and hMSH2:hMSH6 heterodimers. The same regions of the hMSH2monomer interact with regions of both the hMSH3 monomer and the hMSH6monomer. Furthermore, there is a coordinated linear orientation of theseregions, by which is meant that the amino-terminal region of hMSH2associates with the amino-terminal of either hMSH3 or hMSH6 and thecarboxy-terminal region of hMSH2 associates with the carboxy-terminalregion of either hMSH3 or hMSH6. Several missense alterations of hMSH2obtained from HNPCC kindreds were examined and were determined to occurwithin the consensus monomer:monomer interaction regions. None of thesemissense mutations prevented monomer:monomer interaction. These datasupport the idea that an altered interaction of hMSH2 with hMSH3 or analtered interaction of hMSH2 with hMSH6 is unlikely to be causative ofHNPCC.

[0331] In the experiments described in this Example the regions ofmonomer:monomer interaction were ascertained for hMSH2:hMSH3 andhMSH2:hMSH6 heterodimers. Two distinct interaction regions wereidentified for hMSH2:hMSH3 heterodimers and for hMSH2:hMSH6heterodimers. The interaction regions of hMSH2 with either hMSH3 orhMSH6 appeared to be identical. Several missense mutations of hMSH2 wereconstructed. These mutations have been reported by others toco-segregate with HNPCC. None of these alterations affected theinteractions between hMSH2 and either hMSH3 or hMSH6 heterodimers.

[0332] The materials and methods used in the experiments presented inthis Example are not described.

[0333] Reagents and Enzymes

[0334] Restriction endonucleases were obtained from New England Biolabs(Beverly, Mass.). PCR reactions were performed using the High FidelityPCR Kit obtained from Boehringer Mannheim (Mannheim, Germany).Oligonucleotides were synthesized using an Applied Biosystems (FosterCity, Calif.) model 3948 nucleic acid synthesis and purification system.DNA plasmid constructs were purified using Qiagen (Hilden, Germany) DNApurification kits. In vitro transcription and translation (IVTT)reactions were performed using the Promega (Madison, WI) TNTTM CoupledRabbit Reticulocyte Lysate System. Radiolabeled ³⁵S methionine was usedto label proteins and was obtained from Dupont NEN (Wilmington, Del.).Glutathione linked (GST) agarose beads were purchased from Sigma (St.Louis, Mo.).

[0335] Subcloning of hMSH2 and hMSH3

[0336] The cloning of hMSH2, hMSH3, and hMSH6 cDNAs and subcloning intopET expression vectors (obtained from Novagen) has been previouslydescribed (Acharya et al., 1996, Proc. Natl. Acad. Sci. USA93:13629-13634). In this study, we used a HeLa cDNA clone of hMSH3 (GenBank Accession U61981).

[0337] GST fusion proteins were synthesized using the pGEX system(Pharmacia, Sweden). For ease of cloning, plasmid pGEX-4T-2 was modifiedas follows. The vector DNA was digested using EcoRI and BamHIrestriction endonucleases and purified by gel electrophoresis. Adouble-stranded linker oligonucleotide comprising a polynucleotidehaving the nucleotide sequence SEQ ID NO: 13 and a polynucleotide havingthe nucleotide sequence SEQ ID NO: 14 was ligated into the vector. SEQID NO: 13 is 5′-GATCCGAGAA CCTGTACTTC CAGGGACATA TGGCCATGGG TACCG-3′.SEQ ID NO: 14 is 5′-AATTCGGTAC CCATGGCCAT ATGTCCCTGG AAGTACAGGTTCTCG-3′. The vector is herein referred to as pGEX-SGl and permittedsubcloning using NdeI and NcoI restriction endonuclease sites in whichthe ATG initiation codon within each site was in frame with the GSTmoiety. Vector pGEX-SG1 also contained a TEV protease site just upstreamof the NdeI and NcoI sites.

[0338] Construction of hMSH2 truncation mutations

[0339] The hMSH2 deletion mutants were constructed using known PCRtruncation mutagenesis methods. ‘Forward’ primers were generated byadding a polynucleotide homologous with six codons corresponding to thedesired 3′-end of Msh2, starting with a codon having a guanine residuein the 5′-position and adding the 17 nucleotides immediately 3′- withrespect to that residue, to the 3′-end of a polynucleotide having thenucleotide sequence 5′-GCGGATCCCA TGG-3′(SEQ ID NO: 15) ‘Reverse’primers were generated by adding a polynucleotide homologous with the 18nucleotides of the complementary strand corresponding to the six codonsof desired 5′-end of Msh2 to the 3′-end of a polynucleotide having thenucleotide sequence 5′-GGAGGATCCC TA-3′(SEQ ID NO: 16). Using a forwardand reverse primer, a PCR reaction was performed using pET3d-hMSH2 astemplate DNA. The PCR product and pET24d were digested with NcoI andBamHI, purified by gel electrophoresis, and ligated together.

[0340] To make truncated peptides containing an internal deletion, pET24d-hMSH2 (which did not encode amino acid residues 700-800 of hMSH2)was generated by performing PCR on hMSH2 using a pair of polynucleotideprimers having sequences 5′-GCGGATCCCA TGGCAGAAGT GTCCATTGTG-3′(SEQ IDNO: 17) and 5′-GGAGGATCCC ATATGTAGAT TATTAACAGT TGG-3′(SEQ ID NO: 18).The amplification product and pET24d were digested using NcoI and BamHI,and the digested products were purified by gel electrophoresis andligated together. The resulting vector permitted ligation of fragmentsusing NdeI and BamHI. ‘Forward’ primers were designed using the first 18nucleotides of the desired 3′-end of msh2 ligated to the 3′-end of apolynucleotide having the sequence 5′-GGCGGTATCC ATATG-3′(SEQ ID NO:19). The reverse primer was the same as the one described earlier inthis Example. PCR fragments were ligated into this vector using NdeI andBamHl. Site directed mutagenesis of hMSH2 was performed using overlapPCR, as described (Kallal et al., 1997, Mol. Cell. Biol. 17:2897-2907).All of the site directed mutations were completely sequenced using aPerkin Elmer ABI Sequencer with XL upgrade (Perkin Elmer Cetus, Norwalk,Conn.).

[0341] Construction of hMSH3 and hMSH6 truncation mutations

[0342] hMSH3 and hMSH6 truncation constructs were created using a methodanalogous to that used to generate to the hMSH2 deletion mutants.‘Forward’ primers were generated using the same method described fordesigning hMSH2 ‘forward’ primers for hMSH2 mutations havingtruncations. The reverse primers were generated using the same methoddescribed for designing hMSH2 ‘reverse’ primers for hMSH2 mutationshaving either truncations or internal deletions, except that thepolynucleotide had the sequence 5′-GGCATACTCG AGCTA-3′(SEQ ID NO: 20),instead of SEQ ID NO: 16. The PCR amplification product was subclonedinto either pET24d or pGEX-SG1.

[0343] pET24d-hMSH3 (which did not encode amino acid residues 800-990 ofhMSH3) was constructed by performing PCR using msh3 and a pair ofpolynucleotide primers having sequences 5′-GCGGATCCCA TGGATTTTCTAGAGAAATTC-3′(SEQ ID NO: 21) and 5′-GGACGCGTCG TCGACCTAAC CGGTATCTCTGATGAAATAC TC-3′(SEQ ID NO: 22). The amplified product and pET24d weredigested using restriction endonucleases NcoI and SalI and subcloned.This vector permitted ligation of inserts using restrictionendonucleases Agel and XhoI. Forward primers were generated by ligatingsix codons corresponding to the desired 3′-end of msh3 to apolynucleotide having the sequence 5′-GCGGTGACCG GT-3′(SEQ ID NO: 23).Reverse primers were generated as described earlier, only homologouswith the non-coding strand of msh3. PCR was performed, and the amplifiedproducts were ligated.

[0344] In order to avoid errors introduced by random PCR mutagenesis,all PCR amplification products were either completely sequenced or theexperiments were conducted using two separately isolated PCR products.

[0345] GST Fusion Protein Interaction assay

[0346] An overnight culture of E. coli XL-blue cells which harboredpGEX-hMSH(X) (i.e. ‘X’ being 2, 3, or 6) was grown in LB with 50milligrams per milliliter ampicillin. 50 milliliter of Luria brothcontaining ampicillin was inoculated with 1 milliliter of the overnightculture, and the culture was incubated until the optical density, asassessed at 600 nanometers, was about 0.5. IPTG was added to a finalconcentration of 0.1 millimolar, and the culture container placed in ashaker at 30° C. for 2 hours to generate induced cells. Induced cellswere pelleted and resuspended in 800 milliliters of phosphate bufferedsaline (Boehringer Mannheim, Germany) containing protease inhibitors(0.5 millimolar PMSF, 0.8 milligrams per milliliter leupeptin, 0.8milligrams per milliliter pepstatin, and 0. millimolar EDTA). Lysozymewas added to a concentration of 1 milligram per milliliter, and themixture was incubated on ice for 30 minutes. Triton X-100 anddithiothreitol were added to final concentrations of 0.2% (v/v) and 2millimolar, respectively. The lysate was frozen and thawed twice tocompletely lyse the cells. DNase (Boehringer Mannheim, Germany) wasadded to a final concentration of 20 micrograms per milliliter, and thelysate was incubated on ice for an additional 20 minutes. Cell debriswas removed by centrifuging the lysate at 14,000 rpm in a refrigeratedEppendorf (Model 5402) centrifuge for 30 min. The supernatant wastransferred to a new microfuge tube which contained rehydratedGST-agarose beads in a proportion whereby approximately 10-50 nanogramsof protein were present for every 25 microliters of beads that werepresent. GST-fusion protein levels were quantified as described herein.The lysate was incubated with the GST-agarose beads at 4° C. on arocking platform. After rocking for 1-2 hours, the incubation mixturewas centrifuged at 1000 rpm in an Eppendorf microfuge for 30 seconds,the supernatant removed, and the beads were gently resuspended in 500milliliters of Binding Buffer. Binding Buffer consisted of 20 millimolarTris, pH 7.5, 10% (v/v) glycerol, 150 millimolar NaCl, 5 millimolarEDTA, 1 millimolar DTT, 0.1% (v/v) Tween 20, 0.75 milligrams permilliliter BSA, 0.5 millimolar PMSF, 0.8 milligrams per milliliterleupeptin, and 0.8 milligrams per milliliter pepstatin. Thecentrifugation and re-suspension was repeated three times to wash thebeads substantially free of non-specific lysate proteins. Suspendedbeads were added to a 14 milliliter sterile polypropylene tube, dilutedwith Binding Buffer to approximately 50 microliters of packedglutathione beads per milliliter and incubated at 4° C. on a rockingplatform for 30 minutes in order to allow BSA to coat the beads. 500milliliters of these coated GST-fusion protein associated glutathionebeads, which comprised about 10-50 nanograms of bound GST-fusionprotein, was then aliquoted into 1.5 milliliter microfuge tubes.GST-fusion protein expression levels were quantitated by CoomassieBrilliant Blue staining of protein separated by SDS-PAGE gels, using BSAas a standard.

[0347] In vitro transcription and translation (IVTT) reactions involving³⁵S-Methionine were performed with pET-hMSH(Y) (i.e. where ‘Y’ was 2, 3,or 6) using purified DNA according to the manufacturers recommendations.IVTT reactions were pre-run to determine the relative molarconcentration of each construct. This value was calculated using thespecific activity of ³⁵ S-Methionine, correcting for the number ofmethionine residues in each IVTT construct and using SDS-PAGE and aMolecular Dynamics Phosphorlmager device equipped with ImageQuantsoftware (Sunnyvale, Calif.). Up to 10 microliters of the IVTT proteinwas added to each tube such that each sample contained an approximatelyequimolar concentration of IVTT protein. An IVTT reaction which usedpET24d as the vector was added to normalize the total amount of IVTTmixture in each tube. The tubes were incubated for at least one hour at4° C. on a rocker. The beads were washed three times with the BindingBuffer and resuspended in 50 microliters of SDS loading buffer, whichconsisted of 0.25 Tris, pH 6.8, 5% (w/v) sucrose, 2% (w/v) SDS, 5% (v/v)2-mercaptoethanol, and 0.005% (w/v) bromophenol blue. Samples wereresolved by SDS-PAGE, and imaged using the Molecular DynamicsPhosphorlmager (Sunnyvale, Calif.).

[0348] It is recognized that the GST-IVTT interaction assay system isnot quantitative, and may depend on the relative association constant(k_(assoc)) which is related to the concentration of interactingpeptides. Thus, subtle changes in the relative peptide concentrationsmay obscure potentially altered interactions. In order to providecontrol for such concentration-dependent processes between experiments,the molar concentration of the GST-fusion protein and the molarconcentration of the IVTT sample were determined. Furthermore, clearchanges in interaction between hMLH1 and hPMS2 were observed by theinventors using a similar assay system that correlates with alterationsknown to be mutations, rather than polymorphisms.

[0349] The results of the experiments presented in this Example are nowdescribed.

[0350] GST Interaction Assay

[0351] As described elsewhere herein, a physical interaction may bedemonstrated between hMSH2 and either of hMSH3 and hMSH6 usingimmunoprecipitation (IP) reactions with anti-hMSH2 antibodies, whichhave been described in the art and are publicly available. However,interaction-region mapping experiments using truncation mutants of hMSH3and hMSH6 resulted in elevated background as a result of anti-hMSH2antibody binding to the truncated probes. In addition, this IP assay didnot appear sensitive enough to detect weak interactions.

[0352] For these reasons, the alternative assay described herein wasdeveloped. This assay relies on the use of a GST-fusion proteinexpressed in E. coli as a “bait” and in vitro transcribed and translated(IVTT) protein as “prey”. This assay proved to be effective for all ofthe GST-fusion MutS homolog probe combinations used in the studiesdescribed in this application. These GST-fusion MutS homolog probecombinations included GST-hMSH2:IVTT-hMSH3, GST-hMSH3:IVTT-hMSH2,GST-hMSH2:IVTT-hMSH6, and GST-hMSH6:IVTT-hMSH2. The interaction for eachof these IVTT full-length peptides was specific for the correspondingGST-hMSH(X) fusion protein, as evidenced by the observation that nearlyundetectable background non-specific binding was demonstrated byincubation and centrifugal precipitation of the IVTT-MSH(Y) with:

[0353] 1) GST-agarose beads alone;

[0354] 2) E. coli lysate +GST-agarose beads; and

[0355] 3) pGEX (the GST moiety alone) +GST-agarose beads as controls.Furthermore, densitometric comparison of the PAGE lanes containing onlypGEX with PAGE lanes containing GST-hMSH(X) demonstrated that thesignal-to-background ratio in this assay approaches 100. These resultssuggested that this bait-prey system was sufficient to map theinteraction regions of the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers.

[0356] In these studies, a clear interaction between MSH homologs couldbe demonstrated by comparing association of GST alone and IVTT-MSH(Y)with association of GST-MSH(X) and IVTT-MSH(Y), where X and Y areindependently 2, 3, or 6. Furthermore, this assay provided a qualitativemeasure of interaction efficiency, because each experiment contained anearly identical molar ratio of GST-MSH(X) and IVTT-MSH(Y). In addition,the GST-hMSH3 and GST-hMSH6 fusion proteins were demonstrated to beactive for mis-pair binding when they are combined with purified hMSH2.These results indicate that the structure of the hMSH3 and hMSH6proteins is not substantially altered by fusion to GST.

[0357] Interaction Regions of hMSH2 and hMSH3

[0358] The regions of hMSH3 which interact with hMSH2 were determined,as illustrated in FIG. 12. Truncated hMSH3 polypeptides were constructedsuch that the protein was represented by three overlapping polypeptides,as illustrated in FIG. 12, polypeptides 2-4. It was determined thatthere are two separate regions of hMSH3 that interact with hMSH2. It wasrecognized that an amino-terminal region of hMSH3 and a carboxy-terminalregion of hMSH3 are involved in interactions with hMSH2, as illustrated,for example, by the abilities of polypeptides 5 and 10 in FIG. 12 tointeract with GST-hMSH2. The amino-terminal region was determined to belocated within the region of hMSH3 bounded by amino acid residues 126and 250, as indicated by the abilities of polypeptides 6-9 in FIG. 12 tointeract with GST-hMSH2. Because the level of IVTT expression wasinsufficient for polypeptides comprising fewer than one hundred aminoacids, the carboxy-terminal region was mapped using an internal deletionstrategy. Using this strategy, the carboxy-terminal interaction regionwas determined to be located within the region of hMSH3 bounded by aminoacid residues 1050 and 1128, as indicated by the abilities ofpolypeptides 10-14 in FIG. 12 to interact with GST-hMSH2.

[0359] The locations of regions of hMSH2 which interact with hMSH3 weredetermined in a similar fashion. The regions of hMSH2 which interactwith hMSH3 were determined, as illustrated in FIG. 13. Truncated hMSH2polypeptides were constructed such that the protein was represented byfour overlapping polypeptides, as illustrated in FIG. 13, polypeptides2-5. It was determined that hMSH2 comprises two regions which areinvolved in interaction with as indicated by the abilities ofpolypeptides 1-6 in FIG. 13 to interact with GST-hMSH3. Anamino-terminal region was determined to be located within the region ofhMSH2 bounded by amino acid residues 378 and 625, as indicated by theabilities of polypeptides 7-10 in FIG. 13 to interact with GST-hMSH3.The amino acid boundaries of the carboxy-terminal interaction region ofhMSH2 were not resolved with precision, due to sub-optimal signalstrength. Nonetheless, the data illustrated in FIG. 13 indicate that thecarboxy-terminal interaction region of hMSH2 may at least be localizedin the region bounded by amino acid residues 751 and 934 (the carboxyterminus), as indicated by the abilities of polypeptide 6 in FIG. 13 tointeract with GST-hMSH3.

[0360] Because there were two interaction regions between hMSH2 andhMSH3, a system was designed to determine the linear orientation of thetwo regions. GST fusion proteins comprising truncated hMSH3 polypeptideswere constructed. A GST-HMSH3 fusion protein comprising hMSH3 amino acidresidues 1-297 comprised the consensus amino-terminal interactionregion. A GST-hMSH3 fusion protein comprising hMSH3 amino acid residues1025-1128) comprised the consensus carboxy-terminal interaction region.These two fusion proteins were used as “bait” against a series of hMSH2“prey” truncation mutants. We found that non-truncated hMSH2 interactedwith both the GST-hMSH3 fusion protein, as indicated by the ability ofpolypeptide 1 in FIG. 14 to interact with both the GST-hMSH3 fusionprotein comprising the consensus amino-terminal interaction region andthe GST-hMSH3 fusion protein comprising the consensus carboxy-terminalinteraction region. The GST-hMSH3 fusion protein comprising theconsensus amino-terminal interaction region interacted most stronglywith amino acid residues 251-750 of hMSH2 protein, as indicated by theability of polypeptide 4 in FIG. 14 to interact with this GST-hMSH3fusion protein. The GST-hMSH3 fusion protein comprising the consensuscarboxy-terminal interaction region interacted most strongly with aminoacid residues 751-934 of hMSH2 protein, as indicated by the ability ofpolypeptides 5, 6, 7, and 8 in FIG. 14 to interact with this GST-hMSH3fusion protein.

[0361] These results indicate that the amino-terminal interaction regionof hMSH3 normally interacts with the amino-terminal interaction regionof hMSH2 and that the carboxy-region interaction region of hMSH3normally interacts with the carboxyl region interaction region of hMSH2.Use of the GST-hMSH3 fusion protein comprising the consensuscarboxy-terminal interaction region permitted further resolution of thecarboxy-terminal interaction region of hMSH2. It was determined that thecarboxy-terminal interaction region of hMSH2 is bounded by amino acidresidues 875 and 934, as indicated by the ability of polypeptide 8 inFIG. 14 to interact with this GST-hMSH3 fusion protein.

[0362] Interaction Regions of hMSH2 and hMSH6

[0363] Using a similar strategy, the locations of the interactionregions of hMSH2 and hMSH6 were determined. It was recognized that anamino-terminal region of hMSH6 and a carboxy-terminal region of hMSH6are involved in interactions with hMSH2, as illustrated, for example, bythe abilities of polypeptides 1-6 in FIG. 15 to interact with GST-hMSH2.The amino-terminal region was determined to be located within the regionof hMSH6 bounded by amino acid residues 326 and 575, as indicated by theabilities of polypeptides 7-10 in FIG. 15 to interact with GST-hMSH2.The carboxy-terminal region was determined to be located within theregion of hMSH6 bounded by amino acid residues 953 and 1360, asindicated by the abilities of polypeptide 6 to interact with GST-hMSH2.

[0364] The regions of hMSH2 which interact with hMSH6 were determined,as illustrated in FIG. 16. Truncated hMSH2 polypeptides were constructedsuch that the protein was represented by four overlapping polypeptides,as illustrated in FIG. 16, polypeptides 2-5. It was determined thathMSH2 comprises two regions which are involved in interaction withhMSH6, as indicated by the abilities of polypeptides 1-6 in FIG. 15 tointeract with GST-hMSH6. The amino-terminal region was determined to belocated within the region of hMSH2 bounded by amino acid residues 378and 625, as indicated by the abilities of polypeptides 7-10 in FIG. 15to interact with GST-hMSH6. Using a GST fusion protein which contained atruncation mutant of hMSH6 comprising amino acid residues 1302-1360, itwas determined that the carboxyl terminal interaction region of hMSH2 islocated within the region of hMSH2 bounded by amino acid residues 875and 934, as indicated by the ability of polypeptide 8 in FIG. 17 tointeract with this GST fusion protein. The ability of polypeptide 8 inFIG. 17 to interact with this GST fusion protein also indicates that thecarboxy-terminal interaction region of hMSH6 is bounded by amino acidresidues 1302 and 1360.

[0365] These results indicate that the same amino acid regions of hMSH2are involved in the interactions between hMSH2 and hMSH3 and theinteractions between hMSH2 and hMSH6.

[0366] The linear orientation of the hMSH2-hMSH6 interaction regions wasdetermined. Using IVTT amino-terminal and carboxy-terminal hMSH2interaction regions and GST fusion proteins comprising theamino-terminal and carboxy-terminal interaction regions of hMSH6, it wasdetermined that the amino-terminal interaction region of hMSH6 interactswith the amino-terminal interaction region of hMSH2, as indicated by theability of polypeptides 3-5 in FIG. 17 to interact with the GST fusionprotein comprising the amino-terminal interaction region of hMSH6. Itwas furher determined that the carboxy-terminal interaction region ofhMSH6 interacts with the carboxy-terminal interaction region of hMSH2,as indicated by the ability of polypeptides 5-8 in FIG. 17 to interactwith the GST fusion protein comprising the carboxy-terminal interactionregion of hMSH6. Thus, the linear orientation of the interaction regionsof the hMSH2:hMSH6 heterodimer is identical to that of the hMSH2:hMSH3heterodimer.

[0367] Interaction Regions of hMSH2 with Itself

[0368] hMSH2 homodimers bind mismatched duplex DNA (Acharya et al.,1996, Proc. Natl. Acad. Sci. USA 93:13629-13634). Using a GST-hMSH2fusion protein comprising hMSH2 amino acid residues 751-934, it wasdetermined that this portion of hMSH2 (i.e. the carboxy-terminalinteraction region) interacts with the carboxy terminus of hMSH2. Thus,the hMSH2 homodimer exhibits the same carboxy-terminal interactionpattern that was observed between hMSH2 and either of hMSH3 and hMSH6.

[0369] The Effect of hMSH2 Mutations Observed in HNPCC Kindreds onhMSH(X):hMSH(Y) Interaction

[0370] Several HNPCC-associated missense mutations have been describedwhich are located within one of the two interaction regions of hMSH2identified herein. Six of these HNPCC-associated mutations wereconstructed, and the effect of the mutations on hMSH(X):hMSH(Y)interaction were investigated, wherein X and Y are independently 2, 3,or 6. The six HNPCC-associated mutations were those designated L390V,K393M, R524P, N596D, P622L, and T905R. These mutations are described inthe HNPCC database (Peltomalei et al., 1997).

[0371] Interaction experiments were performed using mutated hMSH2fragments which comprised either only an amino-terminal interactionregion or a carboxy-terminal interaction region to eliminate anyconfusion that the presence of multiple interaction regions mightgenerate. These hMSH2 IVTT mutant consensus interaction regions wereexamined for interaction with GST fusion proteins which comprised eitherfull length hMSH3 or full length hMSH6. No difference could be discernedbetween binding of any mutated hMSH2 fragment to either of the fusionproteins and binding of a corresponding wild type hMSH2 fragment toeither of the fusion proteins. These results suggest that alteredinteraction between hMSH2 and either hMSH3 or hMSH6 are not likely to becausative functional defects resulting in HNPCC.

[0372] The results of the experiments described in this Example suggesta model for regional interactions of hMSH2 with hMSH3 and with hMSH6.This model is illustrated in FIG. 18. The results described hereinindicate that hMSH2 employs the same interaction regions, regardless ofwhether it interacts with hMSH3 or with hMSH6. These interactions aremediated by two distinct regions of hMSH2, an amino-terminal interactionregion bounded by amino acid residues 378 and 625 and a carboxy-terminalinteraction region bounded by amino acid residues 875 and 934. Theadenine nucleotide binding region and the putative helix-turn-helixmotif of hMSH2 are not contained within either of these regions. Thus,the results described in this Example indicate that it is unlikely thathelix-turn-helix is essential for interaction of hMSH2 with hMSH3 orwith hMSH6. FIG. 18 illustrates both the relative positions and thelinear orientation of the interaction regions of hMSH2, hMSH3, andhMSH6.

[0373] Since hMSH3 and hMSH6 appear to contact hMSH2 within the samebinding regions, the amino terminal and carboxyl terminal regions ofhMSH3 and hMSH6 were aligned and compared. The amino terminalinteraction regions of hMSH3 and hMSH6 exhibited little identifiablehomology. The carboxyl terminal interaction regions of hMSH3 and hMSH6exhibited moderate homology, 16 of 60 residues being identical. Thecarboxyl-terminal regions of hMSH3 and hMSH6 may provide a conservedfunction for these proteins such as, but not limited to, protein-proteininteraction.

Example 5

[0374] hMSH5, A Human MutS Homolog that Participates in the SecondMeiotic Division

[0375] In the experiments presented in this Example, the human MSH5protein (hMSH5) and the cDNA sequence encoding it are described. Themsh5 gene is located at chromosome 6p22-21, and is involved in meiosis,as evidenced by expression of msh5 in the testes and confinement of suchexpression to secondary spermatocytes and developing spermatids. hMSH5specifically interacts with hMSH4, confirming the generality offunctional heterodimeric interactions in eukaryotic MutS homologs. ThehMSH4:hMSH5 heterodimer may thus be analogized with the hMSH2:hMSH3 andhMSH2:hMSH6 heterodimers.

[0376] The materials and methods described in the experiments presentedin this Example are now described.

[0377] Cloning the hMSH4 and hMSH5 cDNAs

[0378] A search of the NCBI EST database indicated that a 466-base pairsequence derived from Soars human fetal liver spleen cDNA (T67203)exhibited significant homology with both yeast MSH3 and yeast MSH5. Theamino acid sequence of the yeast and the human MSH2 homologs were usedto screen the Human Genome Sciences (HGS, Bethesda, Md.) computerdatabase using TFASTA computer software designed by the Geneticscomputer Group (GCG, University of Wisconsin). The HGS database containsnucleotide sequence information of expressed sequence tags (ESTs) whichidentify a diverse collection of cDNAs derived from more than 400 cDNAlibraries (Adams et al., 1991, Science 252:1651-1656). One EST(designated C4) was determined to exhibit significant homology, but notidentity, to yeast and human MSH2 and MSH3 protein sequences.

[0379] Two PCR fragments were amplified using primers derived from thesetwo EST sequences, which were identified in cDNA derived from humantestis. The PCR product were used to screen a normal human testis cDNAlibrary (obtained from Clontech, Palo Alto, Calif.) using conventionalplaque hybridization techniques. One of the primer sets derived from C4yielded a consistent sequence and identified numerous phage clones. Thisset of primers comprised a forward primer (5′-ACGCCATCTT CACACGAAT-3′;SEQ ID NO: 31) and a reverse primer (5′-TGCAGTGGCA TTGTTCACT-3′; SEQ IDNO: 32). Six clones were identified which were amplified using theseprimers, and these clones were excised using the pDR2 phagemid,according to the manufacturer's recommendations. The six clones weresubcloned into pBSK (Stratagene, La Jolla, Calif.), and double strandsequencing of the six clones was performed using the PRISM™ ReadyReaction DyeDeoxy Terminator Cycle Sequencing Kit and an AppliedBiosystems 377 Sequencer (Foster City, Calif.).

[0380] One clone, designated b29, comprised an open reading frame (ORF)2505 base pairs in length. This ORF comprised one STOP codon N-terminalto the start methionine codon and one STOP codon at a positioncorresponding to the C-terminus of the protein encoded by the ORF. Thecompleteness of the N-terminal region of the ORF was confirmed byperforming a RACE reaction using human normal testis cDNA (Clontech,Palo Alto, Calif.), as described (Apte et al., 1993, BioTechniques15:890-893). The EST sequence obtained from NCBI (T67203) was found tobe located in the C-terminal portion of the b29 ORF.

[0381] Clone b29 was further subcloned into pGEX (Pharmacia, Piscataway,N.J.) for expression of the GST fusion protein in E. coli XL1 Blue(Stratagene, La Jolla, Calif.) and into pET29a (Clontech, Palo Alto,Calif.) for in vitro transcription and translation (IVTT) usingrestriction endonucleases NdeI and NotI (New England Biolabs, Beverley,Ma.).

[0382] An hMSH4 clone was obtained from human testis cDNA (Clontech,Palo Alto, Calif.) by PCR amplification and subsequent ligation into thepCR2.1 vector using a TA cloning kit (Invitrogen, San Diego, Calif.).The primer sequences which were used in these reactions included anouter forward primer (5′-GGAAGGTTTG GGAGGATGC TGAGG-3′; SEQ ID NO: 33),a reverse primer (5′-ATTGTGATTA TTCTTCAGTC TT-3′; SEQ ID NO: 34), anested PCR: forward primer (5′-ATCTCGAGAT GCTGAGGCCT GAG-3′; SEQ ID NO:35), and a second reverse primer (5′-GCGCTAGCTT ATTCTTCAGT CTTTTC-3′;SEQ ID NO: 36). The nucleotide sequence of the amplified clone wasconfirmed by complete double strand sequencing of both strands.

[0383] The hMSH4 clone contained a deletion of a C residue in codon 18and an insertion of a G residue in codon 20, resulting in V19S and V20Smutations. Furthermore, the hMSH4 clone contained a G to A mutation atbase 1219 of the published sequence (numbered starting with the A in theATG initiator codon), which resulted in an E407K amino acidsubstitution. In addition, an apparent polymorphism at codon 368 (CGC toAGA) was detected, which does not alter the coding Arg.

[0384] Chromosomal Mapping of hMSH5

[0385] PCR reactions were performed using the primers described aboverespectively, to screen the GENEBRIDGE-4™ Radiation Hybrid Panel (Hudsonet al., 1995, Science 270:1945-1954). 35 amplification cycles wereperformed using an annealing temperature of 60° C. for 30 secondsfollowed by 72° C. for 1 minute. Fragments were visualized by agarosegel electrophoresis.

[0386] Northern Blotting

[0387] Three multiple tissue northern blots containing poly-A +RNAobtained from a total of 23 different human tissues were obtained fromClontech (Palo Alto, Calif.). 50 nanograms of a full length hMSH5 cDNAand a beta-actin cDNA control were radiolabeled using alpha-(³²P)-dCTPby random primed labeling (Boehringer Mannheim, Germany). Northern Blotswere hybridized according to the manufacturer's instructions. The blotswere washed in 2×SSC containing 0.05% (w/v) SDS at room temperature(i.e. about 20° C.) for a total of 60 minutes and at 50° C. in 0.1×SSC,0.1% (w/v) SDS for atotal of 40 minutes. Phosphorimager screens wereexposed for one day. A 2.5-2.6 kilobase transcript was detected at ahigh level in testis. Tissues with significantly lower expression levelsincluded bone marrow, lymph nodes, brain, and spinal cord.

[0388] Antibodies

[0389] Five different 15-mer peptides were synthesized, eachcorresponding to predicted immunogenic regions of the hMSH5 protein.These peptides were conjugated to hemocyanin, and polyclonal antibodieswere raised in rabbits (H.T.I. Bio-Products, Ramona, Calif.). Antibodyclone C924-2 was found to be most sensitive and specific in Western Blotexperiments and was purified over a Protein-A column for Westernanalysis. Further affinity purification of the antibody was performedusing a crude lysate of SF9 insect cells overexpressing hMSH5 protein.hMSH5 protein lysate was separated by SDS-PAGE, transferred tonitrocellulose and the hMSH5 specific region excised and used toaffinity purify the antibody as described (Wilson et al., 1995, CancerRes. 55:5146-5150).

[0390] Immunohistochemistry

[0391] 5-micron sections of formalin-fixed and paraffin embedded tissueswere cut onto Neoprene coated slides (Aldrich Chemicals, Milwaukee,Wis.). After de-paraffinization, including a 30 minute methanolicperoxide block for endogenous peroxidase activity (Leica Autostainer,Leica, Deerfield, Ill.), the slides were subjected to microwaveradiation in 200 milliliters of Chem.Mate H.I.E.R buffer, pH 5.5-5.7(Ventana Medical Systems, Tucson, Ariz.) at high energy for 5 minutesusing a Panasonic Microwave #NN-5602A (Franklin PK, Ill.). 50milliliters of water were replaced for additional microwave exposure for4 minutes at high energy .

[0392] Immunostaining using the catalyzed signal amplification system(DAKO™, Carpinteria, Calif.) was performed according to themanufacturer's instructions. Incubation with Protein-A and hMSH5specific affinity purified polyclonal antibody was performed for 50minutes at room temperature at concentrations of 1:800 or 1:2000,respectively, using the hMSH2 polyclonal antibody. For counter stainingwith Harris Hematoxylin (Surgipath, Richmond, Ill.), the LeicaAutostainer was used.

[0393] GST Fusion Protein Interaction assay

[0394] 500 microliters taken from a 5 milliliter overnight starterculture of cells which expressed an hMSH2-, hMSH3-, hMSH5-, orhMSH6-pGEX-fusion protein with (or non-fused pGEX as a negative control)was inoculated into 50 milliliters of Luria broth which contained 50micrograms per milliliter ampicillin, and this culture was grown untilthe optical density at 600 nanometers was about 0.5. Protein expressionwas induced by addition of 0.1 millimolar (final concentration) IPTG for2 hours at 30° C. Cells were pelleted and resuspended in 750 microlitersof phosphate buffered saline containing protease inhibitors. A 10 minutedigestion on ice using 1 milligram per milliliter lysozyme was thenperformed. After the addition of 0.2% (v/v) Triton X-100 and 1millimolar dithiothreitol (final concentrations), the lysate wassnap-frozen in liquid nitrogen and thawed twice. DNaseI (200 units permilliliter; Boehringer Mannheim, Germany) digestion was performed usingthe thawed lysate for 30 minutes on ice, after which cell debris wasremoved by centrifugation at 14,000 rpm at 4° C. for 30 minutes in abenchtop microfuge. Equal amounts of lysates obtained from cultureswhich separately expressed one of the fusion proteins (or GST alone as anegative control) were incubated on a rocking platform for 1 hour at 4°C. in the presence of 2 milligrams of glutathione-agarose beads (SigmaChemical Co., St. Louis, Mo.) which had been pre-swollen in phosphatebuffered saline containing protease inhibitors for 1 hour at roomtemperature. The beads were washed three times with 500 microliters ofInteraction Buffer, which comprised 20 millimolar Tris-HCI, pH 7.5, 10%(v/v) Glycerol, 150 millimolar NaCl, 0.1% (v/v) Tween 20, 5 millimolarEDTA, I millimolar DTT, 0.75 milligrams per milliliter bovine serumalbumin (Amresco, Solon, Ohio), and proteinase inhibitors). The beadswere subsequently incubated in Interaction buffer for 1 hour at 4° C. ona rocking platform.

[0395] In vitro transcriptions and translation (IVTT) reactions wereperformed using 1 microgram each of hMSH2, hMSH3, hMSH5, and hMSH6inserts (separately) in pET vectors and using the hMSH4 insert in pCR2.1 using the TNT coupled reticulocyte lysate system (Promega, Madison,Wis.) according to the manufacturer's protocol. About 40 microcuries of³⁵S-methionine was incorporated into each protein. 5 microliters ofindividual IVTTs was added to 500 microliters of glutathione-agarosebeads in Interaction buffer, and the mixture was incubated for 1 hour at4° C. on the rocking platform. After three final washing steps, thesupernatant was removed, and the beads were resuspended in 35microliters of 2× Spear's buffer, boiled for 5 minutes, and centrifugedfor 5 minutes at 14,000 rpm in a benchtop microfuge. 15 microliters ofeach reaction mixture was loaded onto separate lanes of an 8% (w/v)SDS-PAGE Gel (BioRad MiniProtean II), and electrophoresis was performedfor about 90 minutes at 135 volts. Phosphorlmager screens (MolecularDynamics) were exposed to the dried gels for one day.

[0396] The results of the experiments described in this Example are nowdescribed.

[0397] Isolation an chromosomal map of hMSH5, a new human MutS homolog

[0398] Six clones which contained the EST later determined to correspondto msh5 were isolated, and the nucleotide sequence of both strands ofthe clone inserts were determined. Sequence analysis of clone b29indicated the presence of an ORF 2505 base pairs in length. This ORFencoded putative 834-amino-acid protein, as indicated in FIGS. 19A-19C.The predicted molecular weight of the protein is 97 kilodaltons. A STOPcodon was identified beyond the N-terminal end of the ORF, in thenon-coding region, and the completeness of the ORF was confirmed byT-RACE analysis.

[0399] The Genebridge-4 Radiation Hybrid Panel for PCR products having alength corresponding to this ORF. In this way, the msh5 gene was located6.94cR from D6S478 on chromosome 6p22.1-21.3.

[0400] MSH5 defines a new family of MutS homologs involved insporulation and meiosis

[0401] Of all eukaryotic and prokaryotic MutS homologs, the b29 clonewas found to be most closely related to Caenorhabdis elegans MSH5 (29%amino acid identity) and Saccharomyces cerevisiae MSH5 (25% amino acididentity). A region encompassing the adenine nucleotide binding domaindisplayed approximately 60% amino acid identity among these homologues.The gene was therefore designated human msh5.

[0402] Among MutS homologs, the next closest relatives to hMSH5 are theMSH2 proteins. hMSH3 and hMSH6 proteins appear to be less closelyrelated to hMSH5 than are the bacterial MutS proteins. In the presentalignment, the MSH4 proteins appear to be the most divergent of the MutShomologs.

[0403] Expression of hMSH5

[0404] Human msh5 was determined to be transcribed at a high level intestis (FIG. 3). These results correspond to the observation that, inyeast, MSH5 expression was meiosis specific (Hollingsworth et al., 1995,Genes Dev. 9:1728-1739). The size of the human transcript correspondedto the length of the cDNA sequence, which is 2.5 kilobases. The presenceof hMSH5 was detected in testis and tonsil tissue and, at very lowlevels, in two T-and B-cell tumor lines (Jurkat, CEM, Daudi, and GM 1500cell lines) by Western Blot analysis. The Western signal in theseautopsy tissues revealed low molecular weight protein band(s) that werelikely degradation products of the significant autolytic reactionsoccurring in these samples. msh5 expression was also observed in humanbone marrow and lymph node tissues. The presence of msh5 transcript inhuman tissues where B- and T-cells develop as well as expression in theT- and B-cell lines suggests a relationship to cellular developmentprocesses that include recombination events. However, it is alsopossible that the low levels of hMSH5 protein expression in the B- andT-cell lines could result from the fact that the cell lines are derivedfrom hematologic malignancies and thus do not represent normal B- andT-cell precursors or other undefined factors. hMSH5 expression may alsooccur in human brain, spinal cord, and trachea tissues.

[0405] Western analysis suggested that several of the purifiedpolyclonal antibodies derived from synthetic peptides are useful useimmunohistochemical (IHC) studies. IHC stains for surgical specimensobtained from patients with various testicular tumors exhibited nuclearexpression of hMSH5 in spermatids in statu nascendi in round andelongated spermatids (S3). In contrast, all of the preceding phases ofspermatogenesis, as well as the spermatozoa themselves exhibited noexpression of hMSH5. These observations indicate that hMSH5 has aspecific role in the processes associated with the second meioticdivision.

[0406] The testicular histology of the orchiectomy specimens was notentirely normal. Thus, it is possible that hMSH5 was abnormallyexpressed in the testicular samples obtained from surgical patients. Inthe samples examined, histological examination revealed occasionalintratubular neoplasia and the presence of discrete lymphocyticinfiltrates. However, spermatogenesis in these samples was stillfunctioning sufficiently to produce mature sperm cells and a number oftubules were found where there was no evidence of neoplasia.Furthermore, staining of spermatids was evident in all of the tubulesthat appeared normal based on the presence of all stages ofspermatogenesis. Textbook examples of normal tubules would show the celltypes of spermatogenesis filling the entire tubule.

[0407] In contrast, hMSH2 is expressed in the nuclei at nearly allphases of spermatogenesis except for the round and elongated spermatids(where hMSH5 is expressed) and the spermatozoa. Sertoli cells exhibitfaint nuclear staining with hMSH2-specific antibody. hMSH2 expression intissue is clearly correlated with proliferation in general, which isexemplified in the experiments described in this Example by nuclearexpression of hMSH2 in the seminoma. In addition, tissues that werepositive for hMSH2 expression were also positive for expression of theproliferation marker Ki67. hMSH5 protein expression was absent inseminoma and other testicular malignancies such as embryonal cellcarcinoma and mature and immature teratoma. Expression of hMSH5 wasabsent in dividing spermatogonium A, suggesting that expression is notinduced during mitosis.

[0408] Protein Interaction Studies

[0409] Because hMSH2, hMSH3 and hMSH6 are, as described herein, known toact as heterodimers, interaction studies of hMSH5 with hMSH2, hMSH3,hMSH4 and hMSH6 were performed.

[0410] hMSH2 interacts strongly with hMSH3 and hMSH6, as describedherein in Example 1. IVTT-hMSH5 did not interact with GST-hMSH2, -hMSH3or hMSH6 fusion proteins. Similarly, none of IVTF-hMSH2, -hMSH3, and-hMSH6 interacted with GST-hMSH5. The lack of interaction of hMSH5 withhMSH2, hMSH3, and hMSH6 was confirmed as the intensity of the bandsnever exceeded the background. However, there was significantinteraction of GST-hMSH5 with IVTT-hMSH4. Furthermore, a significantinteraction of GST-hMSH3 fusion protein with IVTT-hMSH4 was observed.However, this potential interaction could not be confirmed sincesignificant amounts of soluble GST-hMSH4 fusion protein could not beobtained. These results suggest that hMSH5 specifically interacts withhMSH4 alone.

[0411] In yeast, msh5 mutants have decreased spore viability, increasedlevels of Meiosis I chromosomal nondisjunction and decreased levels ofreciprocal exchange between, but not within, chromosomes (Hollingsworthet al., 1995, Genes Dev. 9:1728-1739). This observation, combined withthe results described herein suggest that hMSH5, and thus also hMSH4, isinvolved in meiotic processing. hMSH5 is located on chromosome 6p22-21and is expressed at very high levels in the testis where meiosis occurscontinually throughout adult life. Immunohistochemical examination oftesticular sections revealed that the protein expression of hMSH5occurred in developing round and elongated spermatids. Spermatogonia andprimary spermatocytes did not express hMSH5, and expression of hMSH5ended abruptly upon development of mature sperm. Because the expressionof hMSH5 is exceedingly strong in the round spermatocytes, it is likelythat expression of hMSH5 begins in the secondary spermatocyte. Theexpression pattern of hMSH5 is consistent with the phenotypes exhibitedin yeast, since the meiosis I chromosomal non-disjunction occurs at thecellular division between the primary and secondary spermatocyte, at thestage where the expression of hMSH5 is likely to be initiated.

[0412] The observations described herein that hMSH5 was expressed inhuman tissues such as bone marrow and lymph nodes, where T-cell andB-cell development takes place, suggests that hMSH5 has a role indevelopment of B-cells, T-cells, or both, and that defects in hMSH5might result in hematological defects.

[0413] hMSH5 appears to specifically interact with hMSH4. No interactionwith hMSH5 above background was observed for hMSH2, hMSH3 or hMSH6.Thus, it is likely that the hMSH4-hMSH5 heterodimer is specific andconstitutes a functional interaction that is separate from hMSH2-hMSH3and hMSH2-hMSH6 heterodimers. Based on the conservation of the adeninenucleotide binding and hydrolysis domain, it is likely that thehMSH4-hMSH5 heterodimer also functions as a molecular switch (Gradia etal., 1997, Cell 91:995-1005).

[0414] The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

[0415] While this invention has been disclosed with reference tospecific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of modifying a mismatched duplex DNA,said method comprising contacting an MSH dimer and said mismatchedduplex DNA in the presence of a binding solution comprising a nucleotideselected from the group consisting of ADP and ATP, wherein theconcentration of ATP in said binding solution is less than about 3micromolar, whereby said MSH dimer associates with the mismatched regionof said mismatched duplex DNA, thereby modifying said mismatched duplexDNA.
 2. The method of claim 1, wherein said MSH dimer is selected fromthe group consisting of a prokaryotic MSH homodimer, a prokaryotic MSHheterodimer, a eukaryotic MSH homodimer, and a eukaryotic MSHheterodimer.
 3. The method of claim 2, wherein said MSH dimer is ahomodimer of a MutS homolog selected from the group consisting of ahuman MutS homolog, a murine MutS homolog, a rat MutS homolog, aDrosophila MutS homolog, a yeast MutS homolog, and a Saccharomycescerevisiae MutS homolog.
 4. The method of claim 2, wherein saideukaryotic MSH homodimer is an MSH2 homodimer.
 5. The method of claim 2,wherein said eukaryotic MSH heterodimer comprises MutS homologsindependently selected from the group consisting of an MSH2 protein, anMSH3 protein, an MSH4 protein, an MSH5 protein, and an MSH6 protein. 6.The method of claim 5, wherein said MSH dimer is selected from the groupconsisting of an MSH2:MSH3 heterodimer, an MSH2:MSH6 heterodimer, and anMSH4:MSH5 heterodimer.
 7. The method of claim 2, wherein saidprokaryotic MSH dimer is a homodimer of Escherichia coli MutS.
 8. Themethod of claim 1, wherein said MSH dimer is substantially purified. 9.The method of claim 1, wherein the concentration of ATP in said bindingsolution is less than about 0.3 micromolar.
 10. The method of claim 9,wherein said binding solution is substantially free of ATP.
 11. Themethod of claim 1, wherein at least one of said MSH dimer and saidmismatched duplex DNA is bound to a support.
 12. The method of claim 1,wherein said mismatched duplex DNA has at least one free end.
 13. Themethod of claim 1, wherein said mismatched duplex DNA comprises a DNAstrand generated by reverse transcription of mRNA obtained from anorganism.
 14. The method of claim 1, wherein said mismatched duplex DNAcomprises a first DNA strand having a reference nucleotide sequence anda second DNA strand selected from the group consisting of a DNA strandobtained from an organism, a DNA strand obtained by amplification of atleast a portion of a polynucleotide obtained from an organism, a DNAstrand obtained by cleavage of a polynucleotide obtained from anorganism, and a DNA strand obtained by reverse transcription of apolynucleotide obtained from an organism.
 15. The method of claim 14,wherein said second DNA strand comprises at least a portion of a geneassociated with a cancer in said organism.
 16. The method of claim 15,wherein said organism is a human and wherein said gene is selected fromthe group consisting of an oncogene and a tumor suppressor gene.
 17. Themethod of claim 16, wherein said gene is selected from the groupconsisting of abl, akt2, apc, bcl2alpha, bcl2beta, bcl3, bcr, brcal,brca2, cbl, ccndl, cdk4, crk-II, csfl r/fms, dbl, dcc, dpc4/smad4,e-cad, e2fl/rbap, egfr/erbb-1, elk1, elk3, eph, erg, etsl, ets2, fer,fgr/src2, fli1/ergb2, fos, fps/fes, fra1, fra2, fyn, hck, hek,her2/erbb-2/neu, her3/erbb-3, her4/erbb-4, hras1, hst2, hstf1, ink4a,ink4b, int2/fgf3, jun, junb, jund, kip2, kit, kras2a, kras2b, 1ck, 1yn,mas, max, mcc, met, m1h1, mos, msh2, msh3, msh6, myb, myba, mybb, myc,myc11, mycn, nf1, nf2, nras, p53, pdgfb, pim1, pms1, pms2, ptc, pten,raf1, rb1, re1, ret, ros1, ski, src1, ta11, tgfbr2, thra1, thrb, tiam1,trk, vav, vh1, wafl, wntl, wnt2, wt1, and yes1.
 18. The method of claim17, wherein said cancer is hereditary non-polyposis colon cancer andsaid gene is selected from the group consisting of m1h1, msh2, msh3,msh6, pms1, and pms2.
 19. The method of claim 15, wherein said cancer isselected from the group consisting of a leukemia, a lymphoma, ameningioma, a mixed tumor of a salivary gland, an adenoma, a carcinoma,an adenocarcinoma, a sarcoma, a dysgerminoma, a retinoblastoma, a Wilms'tumor, a neuroblastoma, a melanoma, and a mesothelioma.
 20. The methodof claim 1, wherein said mismatched duplex DNA and said MSH dimer arecontacted in the presence of at least one non-mismatched duplex DNA. 21.The method of claim 20, further comprising separating said MSH dimerfrom said non-mismatched duplex DNA after contacting said mismatchedduplex DNA and said MSH dimer.
 22. The method of claim 21, furthercomprising dissociating said mismatched duplex DNA and said MSH dimerafter separating said MSH dimer from said non-mismatched duplex DNA andthereafter amplifying said mismatched duplex DNA.
 23. The method ofclaim 22, wherein said MSH dimer is bound to a support prior toseparating said non-mismatched duplex DNA from said MSH dimer.
 24. Themethod of claim 23, wherein said non-mismatched duplex DNA is separatedfrom said MSH dimer in the presence of a separating solution, whereinsaid separating solution is substantially free of ATP.
 25. The method ofclaim 24, further comprising releasing said mismatched duplex DNA fromsaid MSH dimer afier separating said non-mismatched duplex DNA from saidMSH dimer.
 26. The method of claim 25, wherein said mismatched duplexDNA has at least one free end and is released from said MSH dimer bycontacting said MSH dimer with a releasing solution selected from thegroup consisting of a solution comprising ATP and Mg²⁺ ions, a solutioncomprising ATP and a magnesium-chelating agent, a solution comprisinghigh salt, a solution comprising a gamma-modified ATP analog and Mg²⁺ions, and a solution comprising a gamma-hydrolysis-resistant ATP analogand Mg ions.
 27. The method of claim 26, wherein said releasing solutioncomprises ATP and Mg²⁺ ions.
 28. The method of claim 25, wherein saidmismatched duplex DNA does not have a free end and is released from saidMSH dimer by contacting said MSH dimer with a releasing solutionselected from the group consisting of a solution comprising amagnesium-chelating agent, a solution comprising high salt, a solutioncomprising a double-stranded DNA cleaving enzyme, ATP and Mg²⁺ ions, asolution comprising a double-stranded DNA cleaving enzyme, agamma-modified ATP analog, and Mg²⁺ ions, and a solution comprising adouble-stranded DNA cleaving enzyme, a gamma-hydrolysis-resistant ATPanalog, and Mg²⁺ ions.
 29. The method of claim 21, further comprisingcontacting said MSH dimer with a MutL homolog after contacting saidmismatched DNA and said MSH dimer.
 30. The method of claim 1, furthercomprising detecting association of said MSH dimer with said mismatchedduplex DNA.
 31. The method of claim 30, wherein association of said MSHdimer with said mismatched duplex DNA is detected using an assayselected from the group consisting of a gel mobility shift assay, afilter binding assay, an immunological assay, a sedimentationcentrifugation assay, a spectroscopic assay, an optical affinity assay,a DNA footprint assay, and a nucleolytic cleavage protection assay. 32.The method of claim 1, wherein said duplex DNA does not have a free end.33. The method of claim 32, wherein said MSH dimer is present in molarexcess with respect to said mismatched duplex DNA, whereby an average ofmore than one said MSH dimer associates with one molecule of saidmismatched duplex DNA.
 34. A method of modifying a mismatched duplex DNAwhich does not have a free end, said method comprising contacting saidmismatched duplex DNA and an MSH dimer having ADP bound thereto in thepresence of a binding solution, wherein the concentration of ATP in saidbinding solution is less than about 3 micromolar, whereby said homologassociates with the mismatched region of said mismatched duplex DNA,thereby modifying said mismatched duplex DNA.
 35. A method ofsegregating a mismatched duplex DNA from a population of DNA molecules,said method comprising contacting an MSH dimer and said population inthe presence of a binding solution comprising a nucleotide selected fromthe group consisting of ADP and ATP, wherein the concentration of ATP insaid binding solution is less than about 3 micromolar, whereby said MSHdimer associates with said duplex DNA; and segregating said MSH dimerfrom said population, whereby said mismatched duplex DNA is segregatedfrom said population.
 36. A method of detecting a difference between asample nucleotide sequence and a reference nucleotide sequence, saidmethod comprising a) annealing a first DNA strand and a second DNAstrand to form a duplex DNA, i) wherein said first DNA strand has saidsample nucleotide sequence ii) wherein said second DNA strand has anucleotide sequence which is complementary to said reference nucleotidesequence, and iii) whereby if there is a difference between said samplenucleotide sequence and said reference nucleotide sequence then saidduplex DNA is a mismatched duplex DNA; b) thereafter contacting saidduplex DNA and an MSH dimer in the presence of a binding solutioncomprising a nucleotide selected from the group consisting of ADP andATP, wherein the concentration of ATP in said binding solution is lessthan about 3 micromolar, whereby said MSH dimer associates with saidduplex DNA if said duplex DNA is a mismatched duplex DNA; and c)determining whether said MSH dimer is associated with said duplex DNAmolecule, whereby association of said MSH dimer with said duplex DNAmolecule is an indication that there is a difference between said samplenucleotide sequence and said reference nucleotide sequence.
 37. A kitfor separating a mismatched duplex DNA from non-mismatched duplex DNAs,said kit comprising at least two MutS homologs; a linker for bindingsaid at least one of said MutS homologs to a support; and an additionalreagent selected from the group consisting of a nucleotide and areleasing solution, wherein said nucleotide is selected from the groupconsisting of ADP and ATP, and wherein said releasing solution comprisesMg²⁺ and a compound selected from the group consisting of ATP, agamma-modified ATP analog, and a gamma-hydrolysis-resistant ATP analog.38. A method of determining whether a mammal is predisposed forcarcinogenesis, said method comprising a) annealing a first DNA strandand a second DNA strand to form a duplex DNA, p2 i) wherein said firstDNA strand has the nucleotide sequence of at least a portion of a geneselected from the group consisting of an oncogene and a tumor suppressorgene of said mammal, and ii) wherein said second DNA strand has anucleotide sequence which is complementary to the consensus nucleotidesequence of said region, iii) whereby if there is a sequence differencebetween said first DNA strand and said second DNA strand then saidduplex DNA is a mismatched duplex DNA; b) thereafter contacting saidduplex DNA and an MSH dimer in the presence of a binding solutioncomprising a nucleotide selected from the group consisting of ADP andATP, wherein the concentration of ATP in said binding solution is lessthan about 3 micromolar, whereby said MSH dimer associates with saidduplex DNA if said duplex DNA is a mismatched duplex DNA; and c)determining whether said MSH dimer is associated with said duplex DNA,whereby association of said MSH dimer with said duplex DNA is anindication that said mammal is predisposed for carcinogenesis.
 39. Amethod of fractionating a population of duplex DNAs, said methodcomprising a) contacting said population with an MSH dimer in thepresence of a binding solution comprising a nucleotide selected from thegroup consisting of ADP and ATP, wherein the concentration of ATP insaid binding solution is less than about 3 micromolar, whereby said MSHdimer associates with at least one mismatched duplex DNA in saidpopulation; and b) segregating said MSH dimer from said population ofduplex DNAs, whereby said mismatched duplex DNA is segregated from saidpopulation of duplex DNAs, whereby said population is fractionated. 40.A method of selectively amplifying at least one mismatched duplex DNA ofa population of duplex DNAs, said method comprising contacting saidpopulation with an MSH dimer in the presence of a binding solutioncomprising a nucleotide selected from the group consisting of ADP andATP, wherein the concentration of ATP in said binding solution is lessthan about 3 micromolar, whereby said MSH dimer associates with saidmismatched duplex DNA, thereafter segregating said MSH dimer from saidpopulation of duplex DNAs, whereby said mismatched duplex DNA issegregated from said population of duplex DNAs, and thereafteramplifying said mismatched duplex DNA, whereby said mismatched duplexDNA is selectively amplified.
 41. A method of determining whether thenucleotide sequence of a first copy of a genomic sequence differs fromthe nucleotide sequence of a second copy of said genomic sequence, saidmethod comprising amplifying a region of each of said first copy andsaid second copy of said genomic sequence to yield amplified firstcopies and amplified second copies; mixing and denaturing said amplifiedfirst copies and said amplified second copies to form a first mixture;thereafter annealing the nucleic acids in said first mixture to form asecond mixture comprising duplex DNAs, whereby if said the nucleotidesequence of first copy and the nucleotide sequence of said second copyof said genomic sequence differ then at least some of said duplex DNAsare mismatched duplex DNAs; thereafter contacting said second mixturewith an MSH dimer in the presence of a binding solution comprising anucleotide selected from the group consisting of ADP and ATP, whereinthe concentration of ATP in said binding solution is less than about 3micromolar, whereby said MSH dimer associates with said mismatchedduplex DNAs; and determining whether said MSH dimer is associated withat least some of said duplex DNAs, whereby association of said MSH dimerwith said at least some of said duplex DNAs is an indication that thenucleotide sequence of said first copy of said genomic sequence differsfrom the nucleotide sequence of said second copy of said genomicsequence.
 42. A composition for segregating a mismatched duplex DNA froma population of duplex DNAs, said composition comprising an MSHheterodimer bound to a support.
 43. A kit for screening a genomic regionfor a nucleotide sequence which differs from a reference nucleotidesequence, said kit comprising a pair of primers complementary to theends of said region for amplifying said region; a DNA strand having saidreference nucleotide sequence; and at least two MutS homologs.
 44. Anonhuman mammal which is nullizygous for both Msh2 and p53, wherein saidmammal does not express Msh2 or p53, and wherein said mammal exhibits aphenotype selected from the group consisting of inappropriate fetalapoptosis and a predisposition for carcinogenesis.
 45. A method ofmaking a nonhuman mammal which is nullizygous for both Msh2 and p53,does not express Msh2 or p53, and exhibits a phenotype selected from thegroup consisting of a predisposition for inappropriate fetal apoptosisand a predisposition for carcinogenesis, said method comprising matinga) a first parent mammal comprising at least one null allele of Msh2 andat least one null allele of p53 and b) a second parent mammal comprisingat least one null allele of Msh2 and at least one null allele of p53,whereby a nonhuman mammal is generated which is nullizygous for bothMsh2 and p53, does not express Msh2 or p53, and exhibits a phenotypeselected from the group consisting of inappropriate fetal apoptosis anda predisposition for carcinogenesis.
 46. A method of determining whethera compound affects tumorigenesis in mammals, said method comprisingadministering said compound to a first nonhuman mammal which isnullizygous for both Msh2 and p53, does not express Msh2 or p53, andexhibits a predisposition for carcinogenesis, and comparing tumorincidence in said first nonhuman mammal with tumor incidence in a secondnonhuman mammal of the same type which is nullizygous for both Msh2 andp53, does not express Msh2 or p53, exhibits a predisposition forcarcinogenesis, and to which said compound is not administered, wherebya difference in tumor incidence in said first transgenic mammal comparedwith tumor incidence in said second transgenic mammal is an indicationthat said compound affects tumorigenesis in mammals.
 47. A method ofdetermining whether a compound affects a biological phenomenon inmammals, said phenomenon selected from the group consisting ofapoptosis, aging, and fetal development, said method comprisingadministering said compound in utero to a first nonhuman mammalianembryo which is nullizygous for both Msh2 and p53, does not express Msh2or p53, and exhibits a predisposition for inappropriate fetal apoptosis,and comparing the development of said first nonhuman mammalian embryowith the development of a second nonhuman mammalian embryo of the sametype which is nullizygous for both Msh2 and p53, does not express Msh2or p53, exhibits a predisposition for inappropriate fetal apoptosis, andto which said compound is not administered, whereby a difference in thedevelopment of said first nonhuman mammalian embryo compared with thedevelopment of said second nonhuman mammalian embryo is an indicationthat said compound affects said biological phenomenon in mammals.
 48. Acell line which is nullizygous for both Msh2 and p53, does not expressMsh2 or p53, and exhibits a phenotype selected from the group consistingof a predisposition for carcinogenesis and a predisposition forapoptosis, wherein said cell line is made by culturing a cell obtainedfrom the nonhuman mammal of claim
 56. 49. A method of determiningwhether a composition affects expression of a gene selected from thegroup consisting of the p53 gene and a gene encoding a MutS homolog,said method comprising administering said composition to a firstnon-human mammal which is nullizygous for one of said p53 gene and saidgene encoding a MutS homolog; comparing a phenotype of said non-humanmammal with said phenotype of a second non-human mammal of the same typewhich is not nullizygous for said one of said p53 gene and said geneencoding a MutS homolog, wherein said phenotype is selected from thegroup consisting of inappropriate fetal apoptosis and a predispositionfor carcinogenesis; whereby a difference between said phenotype of saidfirst non-human mammal and said phenotype of said second non-humanmammal is an indication that said composition affects expression of theother of said p53 gene and said gene encoding a MutS homolog.
 50. Amethod of determining whether a composition affects expression of a geneselected from the group consisting of the p53 gene and a gene encoding aMutS homolog, said method comprising administering said composition to afirst cell derived from a non-human mammal which is nullizygous for oneof said p53 gene and said gene encoding a MutS homolog; comparing aphenotype of said first cell with said phenotype of a second cellderived from a non-human mammal of the same type which is notnullizygous for said one of said p53 gene and said gene encoding a MutShomolog, wherein said phenotype is selected from the group consisting ofinappropriate fetal apoptosis and a predisposition for carcinogenesis;whereby a difference between said phenotype of said first cell and saidphenotype of said second cell is an indication that said compositionaffects expression of the other of said p53 gene and said gene encodinga MutS homolog.
 51. A composition comprising a human MutS homologfragment, wherein said fragment comprises a MutS homolog interactionregion.
 52. A method of inhibiting association of a first human MutShomolog and a second human MutS homolog, said method comprisingcontacting at least one of said first human MutS homolog and said secondhuman MutS homolog with a human MutS homolog fragment comprising a MutShomolog interaction region, whereby association of said first human MutShomolog and said second human MutS homolog is inhibited.
 53. Acomposition comprising substantially purified hMSH5.
 54. A compositioncomprising an isolated nucleic acid encoding hMSH5.
 55. A method ofmodifying a mismatched duplex DNA, said method comprising contacting anMSH dimer and said mismatched duplex DNA in the presence of a bindingsolution comprising ADP, wherein the concentration of ADP in saidbinding solution is at least about ten times the concentration of ATP,if ATP is present in said binding solution, whereby said MSH dimerassociates with the mismatched region of said mismatched duplex DNA,thereby modifying said mismatched duplex DNA.